RESULTS AND DISCUSSION
4. EFFECTS OF ACTINOBACILUS ACTINOMYCETEMCOMITANS LIPOPOLYSACCHARIDE ON MURINE MACROPHAGES (Study IV)
4.2 Expression of SR-BI and ABCA-1 mRNA
To identify possible mechanisms underlying the LPS-induced cholesterol accumulation into the macrophages, the expression levels of two HDL receptors were determined. Macrophages have several surface receptors, which can mediate efflux of excess cholesterol. SR-BI facilitates the bidirectional efflux of free cholesterol between cells and HDL (Jian et al. 1998), and ABCA-1 mediates the unidirectional efflux of cellular cholesterol and phospholipids to lipid-poor apoA-I (Lawn et al. 1999). JP2- and E. coli -LPS significantly inhibited the ABCA-1 mRNA expression by 59±13% and 71±4%, respectively, compared with control cells with no LPS. None of the LPS preparations had a significant effect on the expression of SR-BI if inhibition of more than 50% was considered significant.
The expression rate of these two transporters in concert with ABCG-1 (Wang et al.
2004) regulates the amount of cholesterol in macrophages (Glass & Witztum 2001). In the present studies, of LPS preparations isolated from A. actinomycetemcomitans, only JP2-LPS inhibited the mRNA expression of ABCA-1 significantly. LPSs from Escherichia coli and Salmonella enterica have earlier been shown to downregulate the mRNA expression levels of
both SR-B1 and ABCA1 as well as protein levels of ABCA1 in mouse macrophages (Baranova et al. 2002, Khovidunkit et al. 2003). Even though differences were present in the ability of the two serotype b strains to inhibit the expression of ABCA1, at least some of the serotype b strains appear to contain LPS that is more atherogenic than LPS isolated from other serotype b strains.
Interestingly, LPS of this particular clone of A. actinomycetemcomitans has the strongest potential to induce proatherogenic effects in macrophages. Further studies with larger numbers of strains are needed to confirm these preliminary results.
LPS isolated from A. actinomycetemcomitans induced IL-1β and TNF-α production by macrophages. LPS preparations of all the strains used in the study induced the production of these cytokines in a time-dependent manner. LPS isolated from IDH781 produced significantly lower levels of TNF-α than LPS isolated from serotype b strains, whereas no differences were seen in the induction of IL-1β between the LPS preparations. Serum LPS concentrations from patients with periodontitis have been shown to correlate with the IL-1β and TNF-α production by macrophages, but the bacterial origin of LPS was not investigated (Pussinen et al. 2004b). A.
actinomycetemcomitans has been demonstrated to induce IL-1β and TNF-α production also in human macrophages (Sosroseno et al. 2002, Kelk et al. 2005).
Based on these results, we conclude that A. actinomycetemcomitans LPS may promote proatherogenic effects by modulating macrophage function. LPS not only enhances foam cell formation but may also impair cholesterol efflux through reduced expression of ABCA-1. Among the LPS preparations isolated from different A. actinomycetemcomitans strains, JP2-LPS representing serotype b was the most effective inducer of foam cell formation and cytokine production and was the only A. actinomycetemcomitans strain suppressing the expression of ABCA-1 significantly. LPS isolated from serotype d strain IDH 781 did not suppress the expression of HDL receptors and induced foam cell formation and cytokine production by macrophages less than serotype b strains. Thus, the proatherogenic potential of A.
actinomycetemcomitans LPS may depend on the infecting A. actinomycetemcomitans clones and correlate with the periodontopathogenic potential of the species.
CONCLUSIONS
This study investigated the dual role of A. actinomycetemcomitans as a pathogen and as a part of the normal oral flora.
A. actinomycetemcomitans serotype b strains were found to grow in elevated proportions in the subgingival flora of patients with periodontitis. This result supports previous findings in which serotype b has been associated with periodontitis. The study further suggests that certain A. actinomycetemcomitans clonotypes have the ability to break the health-compatible homeostasis in the subgingival ecosystem, leading to an overrepresentation of the clonotype.
Natural transformation may be one of the mechanisms in the evolution of the bacteria by which new genetic material, e.g. virulence factors, can be imported to the genome. Natural competence for transformation was observed among A. actinomycetemcomitans serotypes a, d, and e, but any benefit derived from this ability remains unknown and requires further investigation.
A. actinomycetemcomitans Proportion in Proatherogenic potential serotype periodontitis
Serotype b High
Low Serotype c
Intermediate Serotype a
High proatherogenic properties
Intermediate
Serotype d Low proatherogenic
properties Intermediate
Serotype e
Figure 5. Hypothesized relationship between the colonizing clone of A. actinomycetemcomitans and proatherogenic changes in the host.
Serotype d –specific antigen was found to contain the O-antigenic part of lipopolysaccharide. Lipopolysaccharide of A. actinomycetemcomitans was proatherogenic in vitro, and differences were present in proatherogenicity between A. actinomycetemcomitans serotypes. A. actinomycetemcomitans serotype b is the most likely inducer of proatherogenic effects. This is the first study to report variation in the proatherogenic properties of a pathogen.
Interestingly, the same clonotypes of the species are the most likely inducers of both periodontitis and proatherogenic mechanisms (Fig. 5). The proatherogenic structure of LPS is unknown and warrants further study.
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