GA has been utilized as an anti-inflammatory agent for more than two thousand years (4
), and in recent studies inhibited joint destruction in experimental adjuvant-induced autoimmune arthritis (8
). The protective effect of GA on arthritis has been attributed to local increases in GC levels, via inhibition of HSD2 which mediates GC inactivation.
mice have a hyperinflammatory phenotype and are highly susceptible to Pg-
induced alveolar bone destruction (9
). Oral infection of Pg
induces 5 times more alveolar bone loss in IL-10−/−
than in wild type C57BL/6J mice that are resistant to periodontitis (10
). Hence, we chose IL-10−/−
mice as a stringent model to test the therapeutic potential and mechanism of action of GA. As we previously reported, the susceptibility of IL-10−/−
mice to Pg
-induced periodontitis is dependant on a hyperinflammatory state established via IL-12-mediated polarized Th1 immune responses (10
In the present studies we demonstrate that GA completely inhibits Pg-induced bone loss when delivered in either prophylactic or therapeutic regimens, suggesting a mode of action of GA that is exerted at the time of, as well as following periodontal disease initiation. GA also has therapeutic impact in wild-type animals, since it significantly suppressed ligature- and Pg infection-induced alveolar bone loss in wild-type Lewis rats (H.S., unpublished observations). The observation that GA inhibits bone loss in both IL-10−/− and wild type animals clearly indicates that its mode of action is IL-10 independent.
GA has been reported to mediate anti-inflammatory effects in target tissues by increasing active GCs via down-regulation of HSDs (15
). In contrast to autoimmune arthritis (8
), we found that GA failed to down-regulate gingival expression of HSDs, indicating that the HSD-GC axis appears not to be a key mechanism in GA-mediated suppression of alveolar bone loss.
In contrast GA significantly suppressed a number of NF–κB-dependent events including the production of IL-1β and IL-12p70 by LPS-stimulated macrophages (16
LPS was not employed in this study due to its weak and inconsistent effects in this assay system (H.S., unpublished observations). However IL-6, IFNγ and T cell proliferation, all of which are less NF–κB-dependent (18
), were unaffected. Consistent with NF–κB as a primary point of regulation (22
), GA completely inhibited RANKL-stimulated osteoclastogenesis and LPS-induced phosphorylation of NF–κB p105 (NF–κB1) but not NF–κB2 p100/p52 or NF–κB2 p100, under GC-free conditions in vitro
. Taken together, these data strongly indicate that GA-mediated blockade of infection-stimulated alveolar bone loss is dependant on its ability to inhibit the activation of NF–κB, and does not involve modulation of GCs or IL-10.
NFκ –B1 represents the canonical NF-κB pathway whereas NF–κB2 is involved in the non-canonical pathway. The canonical pathway is activated by various stimuli such as cytokines and microbial components. NF–κB1 was also up-regulated in bone marrow cells derived from tail-suspended mice, a model of microgravity in which animals rapidly become osteoporotic (23
). Although Rel-A complexes (Rel-A/c-Rel/p50) will be released from the IκBα inhibitor in the canonical pathway (24
), Rel-A was reported as a key molecule in inhibition of osteolytic osteomyelitis (25
). NF–κB p50/p50 homodimers mediate reactive arthritis, which is an infection-induced inflammation that also involves urethra and eyes (26
). Of interest, in a sepsis arthritis model, systemic inhibition of NF–κB2 using antisense oligonucleotides failed to suppress bone loss (27
). In contrast, non-canonical pathway activation closely relates to autoimmunity, and is not activated by most of the classical NF–κB inducers such as proinflammatory cytokines (28
). In addition to rheumatoid arthritis, NF–κB2 appears to be important in induction of juvenile idiopathic arthritis and psoriatic arthritis (29
). These findings suggest that NF–κB1, but not NF–κB2, may play an important role in infection-stimulated bone loss.
The apparent lack of effect of GA on GC metabolism in periodontitis vs arthritis models may reflect inherent differences in the pathways activated by infection vs an autoimmune disease. HSDs are key regulators of GC pre-receptor metabolism (15
), by interconverting active and inactive GCs. GA preferentially inhibits HSD2, which converts active GCs to their inactive form, but does not affect HSD1, which converts inactive GCs to their active form (5
). HSD2 was elevated in autoimmune arthritis (30
), and was also significantly up-regulated in synovial macrophages of rheumatoid arthritis patients compared to normal synovium (31
). These correlations are consistent with a key role of HSD2 and local GCs in the protective effect of GA in autoimmune arthritis, which is a sterile inflammatory condition. However, direct evidence for the role of this mechanism is still lacking, and as noted earlier, other mechanisms of action have been reported (6
). On the other hand, pro-inflammatory TNFα and IL-1β significantly down-regulate HSD2 in placental trophoblasts (32
), suggesting a GA-independent up-regulatory pathway of active GCs. In contrast, we demonstrate that GA can exert its inhibitory effects via an HSD- and GC-independent mechanism, indicating the complexity of the GA-HSD2-GC axis. Thus, further studies are essential to precisely examine the effect of GA on the NF–κB pathways and HSD-GC axis using NF-κB1−/−