BACKGROUND
periodontitis is a disease affecting the supporting hard and soft tissues of the teeth characterized by loosening and eventual tooth loss  <cit> .
mounting epidemiological evidence has linked periodontitis to systemic illness such as atherosclerosis, and rheumatoid arthritis increasing the morbidity associated with the disease  <cit> .
porphyromonas gingivalis  is a natural member of the oral microflora but can be detected in great numbers in at least 85% of the periodontal lesions.
there is a paradigm shift of the microbiome from health to disease and p. gingivalis has been identified as one of the key anaerobic proteolytic species instrumental in periodontal disease progression  <cit> .
p. gingivalis is a gram negative, assacharolytic, black pigmented bacterium armed with a pleothera of virulence factors such as lipopolysaccharide , gingipains, peptidyl arginine deiminase, haemagglutinins, fimbriae and outer membrane proteins.
these factors are indispensable for the persistence of the organism by enhancing biofilm formation and evading host defense mechanisms  <cit> .
gingipains are typsin-like cysteine proteases that are broadly classified into two main categories –  arginine gingipains  and  lysine gingipain , which can exist in soluble and membrane-bound forms  <cit> .
monocytes and neutrophils are sentinel cells of innate immunity and are found in abundance during periodontal infection  <cit> .
thp- <dig> cells have been widely accepted and used as a surrogate for primary monocytes in biomedical research  <cit> .
toll-like recptors  are germline encoded pattern recognition receptors  present on various cells and they have been evolved to recognize conserved products unique to microbial metabolism and alert the immune system along signaling cascades.
however, in chronic infections like periodontitis, a large number of these pathways converge on a relatively limited set of interaction mechanisms.
polymorphisms of the tlrs have been implicated in various diseases and susceptibility to infections  <cit> .
monocytes have various protease-activated receptors  that are activated by gingipains and other components of the bacteria.
the pars are a unique category of trans-membrane receptors that are activated on the cleavage of the receptor n-terminal part to expose a new, previously cryptic sequence.
the exposed sequence remains tethered to the receptor and acts further as a receptor-activating ligand which results in ca2+ increase and production of cxcl <dig>  <cit> .
it has been shown that platelet activation with gingipains is associated with pars and par <dig> and  <dig> are specifically involved in response to rgp  <cit> .
cxcl <dig> is an important signaling chemokine which is secreted in copious amounts by monocytes in response to infection and it serves to recruit neutrophils to the site of infection along a chemotactic gradient.
stathopolou and colleagues have studied modification of host cytokine responses by p. gingivalis using human gingival epithelial cells  <cit> .
it has been documented that cxcl <dig> exists in two forms:   <dig> aminoacid cxcl <dig> secreted by monocytes and lymphocytes and   <dig> aminoacid cxcl <dig> secreted by various cells of non-immunological origin.
the latter form is broken down into a more potent truncated action, but on prolonged exposure, they are completely inactivated  <cit> .
gingipains have also been known stimulate an innate immune response followed by a potent down regulation of its effects by proteolytic degradation of complement components, anti-bacterial peptides, cytokines and chemokines thereby preventing the resolution of the infection  <cit> .
several studies documenting the effect of purified gingipains or specific gingipain mutants on cells and secretory proteins from the cells have been carried out so far.
previous studies have demonstrated cytokine and chemokine production in thp- <dig> cells and various other cell lines when stimulated with the whole bacterium or components of p. gingivalis  <cit> .
however, the mechanisms involved are still poorly understood.
we hypothesize that, arginine and lysine gingipains differentially regulate cxcl <dig> expression and secretion in monocytes.
the aim of this study is to clarify the role of gingipains in the expression, release and degradation of cxcl <dig> in p. gingivalis- stimulated thp- <dig> cells by using specific kgp or rgp mutants.
methods
cell culture
thp- <dig>  cells were grown in rpmi- <dig> containing 10% fetal bovine serum at 37°c, 5% co <dig>  the logarithmic growth of the cells was maintained between  <dig> ×  <dig> to  <dig> ×  <dig> cells/ml by passage, every 3– <dig> days.
a cell concentration of  <dig> ×  <dig> cells /ml per well was used in a six well plate during each experiment.
bacterial culture and preparation
porphyromonas gingivalis atcc  <dig>  w <dig> and its isogenic mutant strains: arginine gingipain  mutant strain e <dig> and lysine gingipain  mutant strain k1a were kind gifts from dr. m. curtis , were grown in fastidious anaerobic broth .
the bacteria were cultured for  <dig> hours in an anaerobic chamber  containing 80% n <dig>  10% co <dig>  and 10% h <dig> at 37°c.
the bacteria was centrifuged at  <dig> × g for  <dig> minutes at room temperature, washed twice with krebs-ringer glucose buffer  free of calcium  and resuspended in fresh krg buffer without calcium and the concentration was adjusted to  <dig> ×  <dig> cfu/ml.
thirty microlitres of each serial dilution suspensions were plated on fastidious anaerobic agar  plate enriched with 5% defibrinated horse blood and incubated for  <dig> days in the anaerobic chamber for a subsequent colony count.
for heat-killing the bacteria, the prepared bacteria was placed in 70°c heating block for one hour.
to ensure heat-killing, 10μl of this bacterial suspension was streaked on to a fastidious anaerobic agar plate plate enriched with 5% defibrinated horse blood and incubated for  <dig> days in the anaerobic chamber with no observed growth.
in order to stimulate the thp- <dig> cells, a moi   <dig> bacteria was used and incubated for  <dig> hours in a stable environment of 37°c, 5% co <dig> and 95% air.
enzyme-linked immunosorbant assay
elisa was performed on culture supernatant of thp- <dig> cells challenged with the different strains of p. gingivalis to measure cxcl <dig>  after the  <dig> hour treatment, the cell culture suspension was centrifuged at  <dig> × g for  <dig> minutes at room temperature and the cell - free supernatants were aliquoted and stored at -80°c until further analysis using human cxcl <dig> elisa kit .
reverse transcriptase quantitative real-time pcr
the cxcl <dig> expression of thp- <dig> cells in response to the various strains of p. gingivalis was measured using rt-qpcr.
rna isolation was carried out using genejet rna isolation kit  according to manufacturer’s protocol.
reverse transcription was done with equal amounts of rna using cdna synthesis kit .
the primer sequences for cxcl <dig> are as follows: forward sequence: gagagtgattgaga gtggaccac; reverse sequence: cacaaccctctgcacccagttt .
thermal cycling conditions for sybr green  consisted of a denaturation step at 95°c for  <dig> min followed by  <dig> cycles of 95°c for  <dig> s and 60°c for  <dig> s. gene expression was analyzed using a  <dig> ht real-time pcr instrument .
the obtained ct values were normalized against gapdh.
the primer sequences for gapdh are as follows: forward sequence: gtctcctctgacttcaaca gcg; reverse sequence: accaccctgttgctgtagccaa .
all primer sets were specific for their products .
relative quantification of gene-expression was determined by using the ΔΔct method.
the Δct was calculated by subtracting the ct of gapdh from the ct of cxcl <dig> for each sample.
the ΔΔct was calculated by subtracting the Δct of the control sample from the Δct of each treated sample.
fold change was generated by using the equation 2ΔΔct.
cxcl <dig> degradation assay
the cell-free culture supernatants from thp- <dig> cells treated with heat-killed w <dig> were filtered using  <dig>  μm filter to ensure the supernatants were free of bacterial contamination.
the supernatant was diluted  <dig> times using rpmi <dig>  several  <dig> μl aliquots were made and the tubes were incubated with viable or heat killed atcc  <dig> or w <dig> or viable e <dig> and k1a for  <dig> hours at 37°c, 5% co <dig>  three serially diluted concentrations  of viable and heat killed bacteria were used.
after  <dig> hour incubation, the samples were centrifuged at  <dig> x g for  <dig> minutes at room temperature and the supernatant was transferred to a fresh tube and stored at -80°c until further analysis using human cxcl <dig> elisa kit .
endotoxin quantification
the cell culture supernatants of thp- <dig> cells treated with different strains of bacteria were centrifuged at  <dig> × g to get rid of cells, filtered through a  <dig>  μm filter to eliminate bacteria and stored at -80°c until further quantified for endotoxin levels using lal chromogenic endotoxin quantitation kit, according to manufacturer’s instructions.
.
gingipain quantification
the cell culture supernatants of thp- <dig> cells treated with different strains of bacteria were collected and stored at -80°c until further quantified for argine and lysine gingipains using arginine and lysine substrates .
the arginine gingipain substrate peptide sequence is boc-phe-ser -arg-amc  and the lysine gingipain substrate peptide sequence is z-his-glu-lys-amc .
the supernatants  were incubated with either of the substrates at a final concentration of 100μm for one hour at 37°c and the enzyme activity was read in a fluorescence microplate reader  at excitation/emission wavelength settings of 380/  <dig> nm.
scanning laser confocal microscopy
fluorescein isothiocyanate  labeled p. gingivalis was used to stimulate thp- <dig> cells  for  <dig> hours.
the cells were then fixed using 4% ice-cold paraformaldehyde for  <dig> minutes and f-actin visualization was enabled by staining with rhodamine phalloidin .
nucleus was stained using 4', 6-diamidino-2-phenylindole .
thp- <dig> cell and p. gingivalis interaction was analyzed by confocal microscopy .
statistical analyses
the statistically significant differences of data between groups and over time, obtained from real-time qpcr, elisa, lps quantification and cxcl <dig> degradation assay were assessed using one-way anova with bonferroni post-hoc corrections.
all data were represented as mean values with standard deviation.
p-value <  <dig>  was considered significant.
RESULTS
porphyromonas gingivalis induces cxcl <dig> production in thp- <dig> cells
thp- <dig> cells were stimulated with viable or heat-killed p. gingivalis at a moi  <dig> for  <dig> hours.
the cell-culture supernatants were collected and quantified for cxcl <dig> by elisa.
the results show a marked increase in the levels of cxcl <dig>  in p. gingivalis stimulated cells in comparison to unstimulated cells that serve as control.
the highest amounts of cxcl <dig> were observed in the group treated with heat-killed w <dig>  and atcc  <dig>  respectively and this was  <dig> times higher compared to the levels in cells treated with viable atcc  <dig> and w <dig> strains.
among viable w <dig> and its isogenic mutants, k1a induced a significantly higher level of cxcl <dig> compared to w <dig> , whereas e <dig> followed the same trend as viable w <dig> and atcc  <dig>  and was significantly lower than k1a .
porphyromonas gingivalis markedly up-regulates cxcl <dig> in thp- <dig> cells
thp- <dig> cells were stimulated with p. gingivalis for  <dig> hours and mrna was collected and assessed by a quantitative real-time pcr.
the results were normalized to the gapdh gene and the cxcl <dig> gene regulation was estimated in relation to the control.
the expression of cxcl <dig> gene was significantly upregulated by all strains of p. gingivalis except for e <dig> .
the cxcl <dig> gene was accentuated to almost 800-fold in the groups treated with atcc  <dig> and k1a strains .
porphyromonas gingivalis lipopolysaccharide and gingipains are released during incubation with p. gingivalis
lps is an important virulence factor that is capable of stimulating cells in culture  <cit>  and hence the immunogenic nature of lps cannot be overruled.
during the co-incubation of thp- <dig> cells with p. gingivalis, there is release of lps in to the surrounding media.
no lps could be detected in the control as expected.
significant differences were observed when comparing k1a , viable and heat-killed w <dig>  with the control.
the lps obtained on stimulating thp- <dig> cells with e <dig>  was least among the different stimulations and was significantly lower compared to w <dig> .the presence of gingipains in the culture supernatant plays an equally vital role as lps in regulating cxcl <dig>  as expected, supernatants from e <dig> stimulated thp- <dig> cells selectively expressed only kgp activity .
likewise, k1a stimulated thp- <dig> cells expressed only rgp activity.
however, in the atcc <dig> and w <dig> stimulated monocyte suspensions, we detected rgp activity in the culture supernatants and there were significant differences between k1a and the wild-type strain  stimulated monocyte suspensions.
we could not detect kgp activity in the w <dig> or atcc <dig> stimulated thp- <dig> cell co-cultures.
proteolytic efficiency of porphyromonas gingivalis is mainly dependent on arginine gingipain
sterile-filtered supernatants from thp- <dig> cells stimulated with heat-killed w <dig> were incubated with various strains of p. gingivalis in three different concentrations for  <dig> hours to determine cxcl <dig> cleaving efficiency.
all of the viable strains of the two highest concentrations  completely cleaved cxcl <dig>  whereas the heat-killed strains showed no signs of proteolysis irrespective of strain or concentrations.
however, the lowest concentration tested i.e.
<dig> cfu/ml actually demonstrated that arginine gingipain  expresses highest proteolytic activity compared to lysine gingipain  .
variable capacity of p. gingivalis for adhesion and invasion
confocal images of thp- <dig> cells incubated with p. gingivalis for  <dig> hours shows that the different strains of the bacteria adhere to or are inside the thp- <dig> cells .
an antibiotic protection assay after four hour incubation enabled us to retrieve viable p. gingivalis  from the cells .
thp- <dig> cells incubated with viable p. gingivalis  have a tendency to induce morphological changes and formation of large and mixed bacteria/cell aggregates.
stimulation with the heat-killed wild-type strains resulted in larger cells and some of the infected cells were multi-nucleated.
however, thp- <dig> cells stimulated with e <dig> remained mostly in suspension and were morphologically similar to the untreated cells.
discussion
porphyromonas gingivalis is a key periodontopathogen that can seriously debilitate the immune responses and the low grade periodontal infection continuously feeds more bacteria into the blood stream  <cit> .
the bacterial interaction with monocytes through tlrs and pars enables a signalling cascade leading to a release of a plethora of pro-inflammatory mediators e.g.
cxcl <dig>  which is a potent chemokine responsible for neutrophil recruitment  <cit> .
the aim of this study was to elucidate the mechanisms of p. gingivalis interaction with monocytes in terms of induction and expression of cxcl <dig>  by using gingipain mutants, we found that rgp and kgp help p. gingivalis evade the immune system both in an antagonistic and synergistic manner.
the results are consistent with the previous studies and would be a rational to direct future studies using peripheral blood monocytes.
both viable and heat-killed w <dig> strains of p. gingivalis shed significant amounts of lps during interaction with thp- <dig> cells and induced release of cxcl <dig> indicating that the tlrs are involved as lps is heat-stable and is an agonist for tlr <dig> activation.
we observed that the levels of cxcl <dig> are 30– <dig> times higher in thp- <dig> cells stimulated with heat-killed atcc and w <dig> compared to cells exposed to viable bacteria.
tlr <dig> has previously been shown to be activated by both viable and heat-killed p. gingivalis  <cit> .
if the bacteria-induced release of cxcl <dig> is based only on lps, there would have been no difference in cxcl <dig> response between viable and heat- killed bacteria, however, this was not the case.
gingipains are trypsin-like cysteine proteases that can cleave the proteins at arginine and lysine specific sites  <cit> .
we observed that the different strains of p. gingivalis  upregulated cxcl <dig> gene several hundred-folds.
uehara and colleagues investigated the possible synergistic effects of pars, tlrs and nods on cxcl <dig> production in thp- <dig> cells using synthetic peptide ligands and showed that gingipains stimulate the secretion of cytokines from monocytic cells through the activation of pars with synergistic effects by prrs  <cit> .
consequently, the inflammatory response induced by gingipains was not exclusively dependent on their catalytic activity since heat-inactivated bacterial preparations were still effective whereas proteolytic activity was absent by denaturation  <cit> .
the loss of protease activity on heat-treatment was validated by a cytokine degradation functional assay and this also explains the reduced levels of extracellular cxcl <dig> in thp- <dig> cells stimulated with viable bacteria.
measuring the gingipain activity in the supernatant served as a functional validation of the isogenic gingipain mutants as well as the relative expression of arginine and lysine gingipains by the wild-type strains.
aduse-opoku and colleagues showed that rgp and kgp are secreted independent of each other  <cit> .
it is possible that atcc <dig> and w <dig> could be selectively inclined to secreting rgp in aerobic cell culture conditions.
it is evident that the gene transcription patterns do not follow the protein levels and could imply that the post-secretory cxcl <dig> protein is being degraded by gingipains.
the inflammatory character of fimbriae and bacterial dna cannot be disregarded.
thermal inactivation of microorganisms could release bacterial dna due to possible membrane rupture activating tlr <dig> and p. gingivalis fimbriae are also documented to activate tlr <dig> and aid in cxcl <dig> release in human aortic endothelial cells  <cit> .
we show that w <dig> cleaves cxcl <dig> with the same efficiency as k1a, while atcc  <dig> is less effective.
it is clear that there is variation in protease activity among the various wild-type strains of p. gingivalis.
the degradation assay shows that at high titres, both rgp and kgp were equally effective in inactivating cxcl <dig>  whereas at moderate and low titres, rgp was more effective at degrading cxcl <dig>  we have previously shown that cxcl <dig> hydrolysis can be salvaged by using leupeptin which is an arginine-specific gingipain inhibitor  <cit> .
however, all cytokines are not cleaved alike.
stathopolou and colleagues have shown that the rate of il-1β degradation is lower compared to that of il- <dig> or cxcl <dig>  which could be due to the primary, secondary and tertiary molecular structure of il-1β which makes it relatively resistant to hydrolysis by kgp.
it has been shown that interleukin- <dig> is rapidly cleaved by both gingipains, although kgp was found to be more effective  <cit> .
the degradation assay makes it evident that rgp is a more efficient protease at cleaving cxcl <dig>  however, p. gingivalis expressing rgp markedly amplifies cxcl <dig> transcription and significantly higher level of cxcl <dig> was present in culture supernatants of thp- <dig> treated with p. gingivalis expressing only rgp .
this could be due to a complex balance and interaction between induction and degradation.
induction of the cxcl <dig> gene is due to lps, fimbriae, bacterial outer membrane proteins, dna and gingipains, whereas degradation of cxcl <dig> is only protease dependent.
in addition, it has been shown that cxcl <dig> is a secondary cytokine, i.e.
its release could also be mediated by primary cytokines, like il-1β and tnf in an autocrine manner  <cit> .
hence, it is obvious that cxcl <dig> regulation during host pathogen interaction is very elaborate and the in vivo conditions are more dynamic compared to the situation in vitro.
also, gram negative bacteria can shed lps in to the surrounding during cell death, outer membrane vesicle biogenesis and stress  <cit> .
confocal fluorescent images showed that thp- <dig> cells form aggregates when treated with p. gingivalis.
besides the changes in the actin cytoskeleton, p. gingivalis probably increased the expression of cell adhesion molecules in monocytes leading to cell aggregation  <cit> .
thp-1cells stimulated with kgp-deficient k1a appear to be more differentiated and aggregated, compared to the e <dig> -infected thp- <dig> cells, which could be suggestive of a role exhibited by rgps in modulating thp- <dig> cell aggregation and adhesion.
hashizume and colleagues showed that cytoadherence between thp- <dig> cells and endothelial cells were enhanced by treatment with p. gingivalis  <cit> .
p. gingivalis invades or is internalized within the cells.
we were able to culture intracellular bacteria by the antibiotic protection assay after  <dig> hour incubation showing that p. gingivalis survives intracellularly.
the ability of atcc  <dig> to invade endothelial cells has previously been demonstrated in our lab  <cit> .
fimbriae possibly contributes to the intracellular presence of the atcc strain and even sparsely fimbriated strains successfully invaded although in varying degrees indicating there could be other factors supporting adhesion and infection of cells  <cit> .
w <dig> and its mutants possess type iv fima and previous studies have confirmed that the precursor fima can only become mature by the action of rgp and, consequently, the lack of rgp in e <dig> likely renders fima ineffective  <cit> .
the giant multi-nucleated cells that formed on interaction of thp- <dig> cells with viable or heat-killed atcc  <dig> and w <dig> is of additional interest and one could speculate that these giant cells could be of osteoclastic phenotype of importance in the pathogenesis of periodontitis and needs further clarification.
CONCLUSIONS
in conclusion, our results demonstrate that both arginine and lysine gingipains could play a vital role in p. gingivalis-mediated immunomodulation and that rgp appears to be cruciual in the context of cxcl <dig> regulation even at low concentrations.
this study establishes that gingipains are capable of post-secretory chemokine paralysis which would most likely result in abberant neutrophil recruitment to the site of infection and ensuring that a low grade infection would continue unhampered.
the invasion and survival of p. gingivalis within the monocytes could account for the safe passage of the bacteria from the site of periodontal infection to other inflamed sites.
periodontal infections are independent risk factors for several chronic systemic illness and persistent recurrent bacteremia due to denuded oral epithelium results in systemic dissemination of periodontopathogens via the vascular compartment.
competing interests
the authors declare that they have no competing interests.
author’s contributions
tb, hk and kj design the study, kj conducted the experiments.
kj, hk and tb analyzed the data.
kj, hk and tb drafted the article.
all authors read and approved the final manuscript.

 periodontitis is a disease affecting the supporting hard and soft tissues of the teeth characterized by loosening and eventual tooth loss. mounting epidemiological evidence has linked periodontitis to systemic illness such as atherosclerosis, and rheumatoid arthritis increasing the morbidity associated with the disease. porphyromonas gingivalis is a natural member of the oral microflora but can be detected in great numbers in at least 85% of the periodontal lesions. there is a paradigm shift of the microbiome from health to disease and p. gingivalis has been identified as one of the key anaerobic proteolytic species instrumental in periodontal disease progression. p. gingivalis is a gram negative, assacharolytic, black pigmented bacterium armed with a pleothera of virulence factors such as lipopolysaccharide, gingipains, peptidyl arginine deiminase, haemagglutinins, fimbriae and outer membrane proteins. these factors are indispensable for the persistence of the organism by enhancing biofilm formation and evading host defense mechanisms. gingipains are typsin-like cysteine proteases that are broadly classified into two main categories – arginine gingipains and lysine gingipain, which can exist in soluble and membrane-bound forms. monocytes and neutrophils are sentinel cells of innate immunity and are found in abundance during periodontal infection. thp- cells have been widely accepted and used as a surrogate for primary monocytes in biomedical research. toll-like recptors are germline encoded pattern recognition receptors present on various cells and they have been evolved to recognize conserved products unique to microbial metabolism and alert the immune system along signaling cascades. however, in chronic infections like periodontitis, a large number of these pathways converge on a relatively limited set of interaction mechanisms. polymorphisms of the tlrs have been implicated in various diseases and susceptibility to infections. monocytes have various protease-activated receptors that are activated by gingipains and other components of the bacteria. the pars are a unique category of trans-membrane receptors that are activated on the cleavage of the receptor n-terminal part to expose a new, previously cryptic sequence. the exposed sequence remains tethered to the receptor and acts further as a receptor-activating ligand which results in ca2+ increase and production of cxcl. it has been shown that platelet activation with gingipains is associated with pars and par and are specifically involved in response to rgp. cxcl is an important signaling chemokine which is secreted in copious amounts by monocytes in response to infection and it serves to recruit neutrophils to the site of infection along a chemotactic gradient. stathopolou and colleagues have studied modification of host cytokine responses by p. gingivalis using human gingival epithelial cells. it has been documented that cxcl exists in two forms: aminoacid cxcl secreted by monocytes and lymphocytes and aminoacid cxcl secreted by various cells of non-immunological origin. the latter form is broken down into a more potent truncated action, but on prolonged exposure, they are completely inactivated. gingipains have also been known stimulate an innate immune response followed by a potent down regulation of its effects by proteolytic degradation of complement components, anti-bacterial peptides, cytokines and chemokines thereby preventing the resolution of the infection. several studies documenting the effect of purified gingipains or specific gingipain mutants on cells and secretory proteins from the cells have been carried out so far. previous studies have demonstrated cytokine and chemokine production in thp- cells and various other cell lines when stimulated with the whole bacterium or components of p. gingivalis. however, the mechanisms involved are still poorly understood. we hypothesize that, arginine and lysine gingipains differentially regulate cxcl expression and secretion in monocytes. the aim of this study is to clarify the role of gingipains in the expression, release and degradation of cxcl in p. gingivalis- stimulated thp- cells by using specific kgp or rgp mutants. methods cell culture thp- cells were grown in rpmi- containing 10% fetal bovine serum at 37°c, 5% co the logarithmic growth of the cells was maintained between × to × cells/ml by passage, every 3– days. a cell concentration of × cells /ml per well was used in a six well plate during each experiment. bacterial culture and preparation porphyromonas gingivalis atcc w and its isogenic mutant strains: arginine gingipain mutant strain e and lysine gingipain mutant strain k1a were kind gifts from dr. m. curtis, were grown in fastidious anaerobic broth. the bacteria were cultured for hours in an anaerobic chamber containing 80% n 10% co and 10% h at 37°c. the bacteria was centrifuged at × g for minutes at room temperature, washed twice with krebs-ringer glucose buffer free of calcium and resuspended in fresh krg buffer without calcium and the concentration was adjusted to × cfu/ml. thirty microlitres of each serial dilution suspensions were plated on fastidious anaerobic agar plate enriched with 5% defibrinated horse blood and incubated for days in the anaerobic chamber for a subsequent colony count. for heat-killing the bacteria, the prepared bacteria was placed in 70°c heating block for one hour. to ensure heat-killing, 10μl of this bacterial suspension was streaked on to a fastidious anaerobic agar plate plate enriched with 5% defibrinated horse blood and incubated for days in the anaerobic chamber with no observed growth. in order to stimulate the thp- cells, a moi bacteria was used and incubated for hours in a stable environment of 37°c, 5% co and 95% air. enzyme-linked immunosorbant assay elisa was performed on culture supernatant of thp- cells challenged with the different strains of p. gingivalis to measure cxcl after the hour treatment, the cell culture suspension was centrifuged at × g for minutes at room temperature and the cell - free supernatants were aliquoted and stored at -80°c until further analysis using human cxcl elisa kit. reverse transcriptase quantitative real-time pcr the cxcl expression of thp- cells in response to the various strains of p. gingivalis was measured using rt-qpcr. rna isolation was carried out using genejet rna isolation kit according to manufacturer’s protocol. reverse transcription was done with equal amounts of rna using cdna synthesis kit. the primer sequences for cxcl are as follows: forward sequence: gagagtgattgaga gtggaccac; reverse sequence: cacaaccctctgcacccagttt. thermal cycling conditions for sybr green consisted of a denaturation step at 95°c for min followed by cycles of 95°c for s and 60°c for s. gene expression was analyzed using a ht real-time pcr instrument. the obtained ct values were normalized against gapdh. the primer sequences for gapdh are as follows: forward sequence: gtctcctctgacttcaaca gcg; reverse sequence: accaccctgttgctgtagccaa. all primer sets were specific for their products. relative quantification of gene-expression was determined by using the ΔΔct method. the Δct was calculated by subtracting the ct of gapdh from the ct of cxcl for each sample. the ΔΔct was calculated by subtracting the Δct of the control sample from the Δct of each treated sample. fold change was generated by using the equation 2ΔΔct. cxcl degradation assay the cell-free culture supernatants from thp- cells treated with heat-killed w were filtered using μm filter to ensure the supernatants were free of bacterial contamination. the supernatant was diluted times using rpmi several μl aliquots were made and the tubes were incubated with viable or heat killed atcc or w or viable e and k1a for hours at 37°c, 5% co three serially diluted concentrations of viable and heat killed bacteria were used. after hour incubation, the samples were centrifuged at x g for minutes at room temperature and the supernatant was transferred to a fresh tube and stored at -80°c until further analysis using human cxcl elisa kit. endotoxin quantification the cell culture supernatants of thp- cells treated with different strains of bacteria were centrifuged at × g to get rid of cells, filtered through a μm filter to eliminate bacteria and stored at -80°c until further quantified for endotoxin levels using lal chromogenic endotoxin quantitation kit, according to manufacturer’s instructions.. gingipain quantification the cell culture supernatants of thp- cells treated with different strains of bacteria were collected and stored at -80°c until further quantified for argine and lysine gingipains using arginine and lysine substrates. the arginine gingipain substrate peptide sequence is boc-phe-ser -arg-amc and the lysine gingipain substrate peptide sequence is z-his-glu-lys-amc. the supernatants were incubated with either of the substrates at a final concentration of 100μm for one hour at 37°c and the enzyme activity was read in a fluorescence microplate reader at excitation/emission wavelength settings of 380/ nm. scanning laser confocal microscopy fluorescein isothiocyanate labeled p. gingivalis was used to stimulate thp- cells for hours. the cells were then fixed using 4% ice-cold paraformaldehyde for minutes and f-actin visualization was enabled by staining with rhodamine phalloidin. nucleus was stained using 4', 6-diamidino-2-phenylindole. thp- cell and p. gingivalis interaction was analyzed by confocal microscopy. statistical analyses the statistically significant differences of data between groups and over time, obtained from real-time qpcr, elisa, lps quantification and cxcl degradation assay were assessed using one-way anova with bonferroni post-hoc corrections. all data were represented as mean values with standard deviation. p-value <  was considered significant.  porphyromonas gingivalis induces cxcl production in thp- cells thp- cells were stimulated with viable or heat-killed p. gingivalis at a moi for hours. the cell-culture supernatants were collected and quantified for cxcl by elisa. the results show a marked increase in the levels of cxcl in p. gingivalis stimulated cells in comparison to unstimulated cells that serve as control. the highest amounts of cxcl were observed in the group treated with heat-killed w and atcc respectively and this was times higher compared to the levels in cells treated with viable atcc and w strains. among viable w and its isogenic mutants, k1a induced a significantly higher level of cxcl compared to w, whereas e followed the same trend as viable w and atcc and was significantly lower than k1a. porphyromonas gingivalis markedly up-regulates cxcl in thp- cells thp- cells were stimulated with p. gingivalis for hours and mrna was collected and assessed by a quantitative real-time pcr. the results were normalized to the gapdh gene and the cxcl gene regulation was estimated in relation to the control. the expression of cxcl gene was significantly upregulated by all strains of p. gingivalis except for e. the cxcl gene was accentuated to almost 800-fold in the groups treated with atcc and k1a strains. porphyromonas gingivalis lipopolysaccharide and gingipains are released during incubation with p. gingivalis lps is an important virulence factor that is capable of stimulating cells in culture and hence the immunogenic nature of lps cannot be overruled. during the co-incubation of thp- cells with p. gingivalis, there is release of lps in to the surrounding media. no lps could be detected in the control as expected. significant differences were observed when comparing k1a, viable and heat-killed w with the control. the lps obtained on stimulating thp- cells with e was least among the different stimulations and was significantly lower compared to w.the presence of gingipains in the culture supernatant plays an equally vital role as lps in regulating cxcl as expected, supernatants from e stimulated thp- cells selectively expressed only kgp activity. likewise, k1a stimulated thp- cells expressed only rgp activity. however, in the atcc and w stimulated monocyte suspensions, we detected rgp activity in the culture supernatants and there were significant differences between k1a and the wild-type strain stimulated monocyte suspensions. we could not detect kgp activity in the w or atcc stimulated thp- cell co-cultures. proteolytic efficiency of porphyromonas gingivalis is mainly dependent on arginine gingipain sterile-filtered supernatants from thp- cells stimulated with heat-killed w were incubated with various strains of p. gingivalis in three different concentrations for hours to determine cxcl cleaving efficiency. all of the viable strains of the two highest concentrations completely cleaved cxcl whereas the heat-killed strains showed no signs of proteolysis irrespective of strain or concentrations. however, the lowest concentration tested i.e. cfu/ml actually demonstrated that arginine gingipain expresses highest proteolytic activity compared to lysine gingipain. variable capacity of p. gingivalis for adhesion and invasion confocal images of thp- cells incubated with p. gingivalis for hours shows that the different strains of the bacteria adhere to or are inside the thp- cells. an antibiotic protection assay after four hour incubation enabled us to retrieve viable p. gingivalis from the cells. thp- cells incubated with viable p. gingivalis have a tendency to induce morphological changes and formation of large and mixed bacteria/cell aggregates. stimulation with the heat-killed wild-type strains resulted in larger cells and some of the infected cells were multi-nucleated. however, thp- cells stimulated with e remained mostly in suspension and were morphologically similar to the untreated cells. discussion porphyromonas gingivalis is a key periodontopathogen that can seriously debilitate the immune responses and the low grade periodontal infection continuously feeds more bacteria into the blood stream. the bacterial interaction with monocytes through tlrs and pars enables a signalling cascade leading to a release of a plethora of pro-inflammatory mediators e.g. cxcl which is a potent chemokine responsible for neutrophil recruitment. the aim of this study was to elucidate the mechanisms of p. gingivalis interaction with monocytes in terms of induction and expression of cxcl by using gingipain mutants, we found that rgp and kgp help p. gingivalis evade the immune system both in an antagonistic and synergistic manner. the results are consistent with the previous studies and would be a rational to direct future studies using peripheral blood monocytes. both viable and heat-killed w strains of p. gingivalis shed significant amounts of lps during interaction with thp- cells and induced release of cxcl indicating that the tlrs are involved as lps is heat-stable and is an agonist for tlr activation. we observed that the levels of cxcl are 30– times higher in thp- cells stimulated with heat-killed atcc and w compared to cells exposed to viable bacteria. tlr has previously been shown to be activated by both viable and heat-killed p. gingivalis. if the bacteria-induced release of cxcl is based only on lps, there would have been no difference in cxcl response between viable and heat- killed bacteria, however, this was not the case. gingipains are trypsin-like cysteine proteases that can cleave the proteins at arginine and lysine specific sites. we observed that the different strains of p. gingivalis upregulated cxcl gene several hundred-folds. uehara and colleagues investigated the possible synergistic effects of pars, tlrs and nods on cxcl production in thp- cells using synthetic peptide ligands and showed that gingipains stimulate the secretion of cytokines from monocytic cells through the activation of pars with synergistic effects by prrs. consequently, the inflammatory response induced by gingipains was not exclusively dependent on their catalytic activity since heat-inactivated bacterial preparations were still effective whereas proteolytic activity was absent by denaturation. the loss of protease activity on heat-treatment was validated by a cytokine degradation functional assay and this also explains the reduced levels of extracellular cxcl in thp- cells stimulated with viable bacteria. measuring the gingipain activity in the supernatant served as a functional validation of the isogenic gingipain mutants as well as the relative expression of arginine and lysine gingipains by the wild-type strains. aduse-opoku and colleagues showed that rgp and kgp are secreted independent of each other. it is possible that atcc and w could be selectively inclined to secreting rgp in aerobic cell culture conditions. it is evident that the gene transcription patterns do not follow the protein levels and could imply that the post-secretory cxcl protein is being degraded by gingipains. the inflammatory character of fimbriae and bacterial dna cannot be disregarded. thermal inactivation of microorganisms could release bacterial dna due to possible membrane rupture activating tlr and p. gingivalis fimbriae are also documented to activate tlr and aid in cxcl release in human aortic endothelial cells. we show that w cleaves cxcl with the same efficiency as k1a, while atcc is less effective. it is clear that there is variation in protease activity among the various wild-type strains of p. gingivalis. the degradation assay shows that at high titres, both rgp and kgp were equally effective in inactivating cxcl whereas at moderate and low titres, rgp was more effective at degrading cxcl we have previously shown that cxcl hydrolysis can be salvaged by using leupeptin which is an arginine-specific gingipain inhibitor. however, all cytokines are not cleaved alike. stathopolou and colleagues have shown that the rate of il-1β degradation is lower compared to that of il- or cxcl which could be due to the primary, secondary and tertiary molecular structure of il-1β which makes it relatively resistant to hydrolysis by kgp. it has been shown that interleukin- is rapidly cleaved by both gingipains, although kgp was found to be more effective. the degradation assay makes it evident that rgp is a more efficient protease at cleaving cxcl however, p. gingivalis expressing rgp markedly amplifies cxcl transcription and significantly higher level of cxcl was present in culture supernatants of thp- treated with p. gingivalis expressing only rgp. this could be due to a complex balance and interaction between induction and degradation. induction of the cxcl gene is due to lps, fimbriae, bacterial outer membrane proteins, dna and gingipains, whereas degradation of cxcl is only protease dependent. in addition, it has been shown that cxcl is a secondary cytokine, i.e. its release could also be mediated by primary cytokines, like il-1β and tnf in an autocrine manner. hence, it is obvious that cxcl regulation during host pathogen interaction is very elaborate and the in vivo conditions are more dynamic compared to the situation in vitro. also, gram negative bacteria can shed lps in to the surrounding during cell death, outer membrane vesicle biogenesis and stress. confocal fluorescent images showed that thp- cells form aggregates when treated with p. gingivalis. besides the changes in the actin cytoskeleton, p. gingivalis probably increased the expression of cell adhesion molecules in monocytes leading to cell aggregation. thp-1cells stimulated with kgp-deficient k1a appear to be more differentiated and aggregated, compared to the e -infected thp- cells, which could be suggestive of a role exhibited by rgps in modulating thp- cell aggregation and adhesion. hashizume and colleagues showed that cytoadherence between thp- cells and endothelial cells were enhanced by treatment with p. gingivalis. p. gingivalis invades or is internalized within the cells. we were able to culture intracellular bacteria by the antibiotic protection assay after hour incubation showing that p. gingivalis survives intracellularly. the ability of atcc to invade endothelial cells has previously been demonstrated in our lab. fimbriae possibly contributes to the intracellular presence of the atcc strain and even sparsely fimbriated strains successfully invaded although in varying degrees indicating there could be other factors supporting adhesion and infection of cells. w and its mutants possess type iv fima and previous studies have confirmed that the precursor fima can only become mature by the action of rgp and, consequently, the lack of rgp in e likely renders fima ineffective. the giant multi-nucleated cells that formed on interaction of thp- cells with viable or heat-killed atcc and w is of additional interest and one could speculate that these giant cells could be of osteoclastic phenotype of importance in the pathogenesis of periodontitis and needs further clarification.  in conclusion, our results demonstrate that both arginine and lysine gingipains could play a vital role in p. gingivalis-mediated immunomodulation and that rgp appears to be cruciual in the context of cxcl regulation even at low concentrations. this study establishes that gingipains are capable of post-secretory chemokine paralysis which would most likely result in abberant neutrophil recruitment to the site of infection and ensuring that a low grade infection would continue unhampered. the invasion and survival of p. gingivalis within the monocytes could account for the safe passage of the bacteria from the site of periodontal infection to other inflamed sites. periodontal infections are independent risk factors for several chronic systemic illness and persistent recurrent bacteremia due to denuded oral epithelium results in systemic dissemination of periodontopathogens via the vascular compartment. competing interests the authors declare that they have no competing interests. author’s contributions tb, hk and kj design the study, kj conducted the experiments. kj, hk and tb analyzed the data. kj, hk and tb drafted the article. all authors read and approved the final manuscript. 
this study evaluated the interaction between p. gingivalis and thp- cells, a widely used monocytic cell line, in vitro with a focus on cxcl at the gene and protein levels and its fate thereafter in cell culture supernatants. elisa and qpcr results show an elevated cxcl expression and secretion in thp- cells in response to p. gingivalis, where the heat-killed atcc and w induced higher levels of cxcl in comparison to their viable counterparts. of all tested p. gingivalis strains, adhesion and internalization in thp- cells was least conspicuous by rgp-deficient p. gingivalis, as demonstrated by confocal imaging. thp- cells were stimulated with viable and heat-killed wild-type strains atcc or w or viable isogenic gingipain mutants of w e or k1a, for hours. the wild-type strains secreted relatively more rgp during the co-culture with thp- cells. furthermore, the kgp-deficient mutant k1a caused a higher cxcl response compared to the rgp-deficient e chromogenic quantification of lipopolysaccharide in supernatant showed no significant differences between viable and heat killed bacteria except that w shed highest levels of lps. porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. porphyromonas gingivalisthp- cellsgingipainsmutantscxcl degradation the bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains and lysine gingipain.
 porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. the bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains and lysine gingipain. this study evaluated the interaction between p. gingivalis and thp- cells, a widely used monocytic cell line, in vitro with a focus on cxcl at the gene and protein levels and its fate thereafter in cell culture supernatants. thp- cells were stimulated with viable and heat-killed wild-type strains atcc or w or viable isogenic gingipain mutants of w e or k1a, for hours.  elisa and qpcr results show an elevated cxcl expression and secretion in thp- cells in response to p. gingivalis, where the heat-killed atcc and w induced higher levels of cxcl in comparison to their viable counterparts. furthermore, the kgp-deficient mutant k1a caused a higher cxcl response compared to the rgp-deficient e chromogenic quantification of lipopolysaccharide in supernatant showed no significant differences between viable and heat killed bacteria except that w shed highest levels of lps. the wild-type strains secreted relatively more rgp during the co-culture with thp- cells. the cxcl degradation assay of filter-sterilized supernatant from heat-killed w treated cells showed that rgp was most efficient at cxcl hydrolysis. of all tested p. gingivalis strains, adhesion and internalization in thp- cells was least conspicuous by rgp-deficient p. gingivalis, as demonstrated by confocal imaging.  w and its kgp mutant k1a exhibit a higher immunogenic and proteolytic function in comparison to the rgp mutant e since k1a differs from e in the expression of rgp, it is rational to conclude that rgp contributes to immunomodulation in a more dynamic manner in comparison to kgp. also, w is a more virulent strain when compared to the laboratory strain atcc porphyromonas gingivalisthp- cellsgingipainsmutantscxcl degradation 