Previous studies have implicated MMP-9 as playing an important role in the dissemination of B. burgdorferi
during the course of infection. In these experiments, we report that MMP-9 was not required for the dissemination of the spirochete since the time to dissemination to distant sites and the spirochetal burden in the hearts and ankle joints of MMP-9−/−
mice were similar to those found in MMP-9+/+
mice. There was a slight trend toward earlier dissemination in the WT mice versus the MMP-9−/−
mice. Gebbia et al. (27
) have previously shown that MMP-9 could enhance penetration through ECM components in vitro. Thus, while we cannot rule out that MMP-9 plays some role in dissemination, it is clearly not required. It is certainly possible that MMP-9 is one of a number of different but redundant proteases utilized by B. burgdorferi
in order to digest ECM proteins to spread from the site of inoculation. B. burgdorferi
binds plasmin, which is also capable of digesting ECM proteins directly, as well as activating MMP-9 and other MMPs. In vitro, plasmin increases penetration of B. burgdorferi
through ECM proteins by direct digestion and by activation of other MMPs (16
). Studies in plasmin(ogen)-deficient mice also did not show an effect on dissemination of the organism to distant organs, although there was a slight effect on bacteremia (15
In spite of similar spirochetal tissue burdens, the MMP-9−/− mice showed significantly reduced swelling and pathology in the ankle joints compared to the WT controls. Because the mice were backcrossed to the arthritis-susceptible C3H background from a resistant C57BL/6 background, there is a small chance that a retained gene from C57BL/6 accounts for the lack of arthritis. However, this is very unlikely with 10 backcrosses. In addition, the proinflammatory chemokine and cytokine induction by B. burgdorferi were similar between the two groups of mice, suggesting that the signals to attract inflammatory cells to the areas of B. burgdorferi localization are similar in the WT and KO mice. There are several potential explanations for why inflammation may be decreased in MMP-9−/− mice despite similar levels of inflammatory cytokines.
One possibility is that MMP-9 is utilized by inflammatory cells to break down ECM barriers and follow chemotactic gradients to areas where the organism has localized. In this scenario, the lack of MMP-9 would result in inflammatory cells being unable to respond to chemoattractive signals due to their inability to degrade and maneuver through the ECM. There are some data to suggest that MMP-9-mediated degradation of the ECM is essential for the immune effector cells to extravasate to the site of infection (21
). In the absence of this degradation, cellular honing to the site of infection does not occur, and inflammation is consequently dampened. However, this remains controversial since other studies have reported different results for the requirement of MMP-9 ECM degradation in this process. These studies showed similar movement of effector cells in response to either IL-8 or murine TNF-α in spite of the absence of MMP-9 (3
). With B. burgdorferi
infection, we observed an effect of MMP-9 deficiency on inflammation in the joint but not in the heart. This suggests to us that the ability of inflammatory cells to migrate was not compromised in the absence of MMP-9. However, we cannot rule out the possibility that MMP-9 is required for migration into the joint space but not the heart due to differences in tissue composition. However, since neither the joint space or cardiac tissue present specific barriers to cell migration through the endothelium, it is less likely that any tissue differences account for a difference in migration.
An alternative possibility is that the proteolytic activity of MMP-9 is required to activate an important inflammatory factor. MMP-9 cleavage of certain chemokines has been shown to increase their potency as proinflammatory molecules. For example, truncation of the amino-terminal end of IL-8 by MMP-9 potentiates its chemotactic properties by tenfold and the mouse neutrophilic chemoattractant GCP-2/LIX by twofold (52
). MMP-9 can also change the proinflammatory environment created by specific cytokines. Activation of IL-1β, a potent proinflammatory cytokine, from its dormant precursor by MMP-9 also potentiates the innate immune response through the removal of its activity-blocking prodomain (46
). Similarly, TNF-α is proteolytically cleaved from its 233-amino-acid anchored form to its 157-amino-acid activated soluble form (25
). Increases in potency of chemokines through cleavage and activation by MMP-9 would not be detected by the transcriptional PCR analysis we performed. Thus, it is possible that, in MMP-9−/−
mice, a similar profile of proinflammatory chemokines and cytokines is upregulated by the presence of the organism; however, without further processing by MMP-9, many of the inflammatory molecules may not be fully activated.
In addition to directly cleaving chemokines and cytokines, MMP-9 also can control inflammation by controlling the release of other soluble inflammatory mediators that are usually bound to the ECM. Once released from the ECM, these mediators then create a concentration gradient that can attract the innate immune effector cells to the site of infection. An example of this type of MMP-9 inflammatory regulation involves the release of proinflammatory proteoglycans from the ECM. Upon stimulation with CXCL-12, HeLa cells upregulate MMP-9, which in turn can cause the release of specific proteoglycans called syndecans (11
). These shed syndecans have been shown to mediate inflammation (23
Recent research has revealed that the components of the ECM do not merely provide scaffolding for other cells but can in fact play a direct role in immune modulation. Fragments of degraded ECM components have been shown to mediate inflammation by serving as chemotactic peptides that attract innate immune effector cells to the site of infection (2
). Collagen, elastins, laminins, and fibronectins which are all substrates of MMP-9, have been shown to become chemotactic after they are degraded by various proteases (including bacterial collegenase, human neutrophil elastase, MMP-2, and thermolysin), with the resulting peptide fragments having chemotactic properties for both neutrophils and macrophages (1
). This was suggested as a possible mechanism for the decreased inflammation seen in MMP-9−/−
mice challenged with F. tularensis
mice showed lower levels of the neutrophil proinflammatory tripeptide Pro-Gly-Pro, which is a by-product of ECM degradation by MMP-9 (40
To explore the hypothesis that MMP-9 may be degrading components of the ECM, and collagen more specifically, we digested various forms of collagen with MMP-9 and tested whether these fragments could cause the chemotaxis of human PBMC and potentially play a role in the progression of Lyme arthritis. We found that degraded type I collagen and potentially type IV collagen were chemotactic after digestion with MMP-9. To our knowledge, this is the first report of MMP-9 cleavage of collagen resulting in the development of a chemotactic molecule(s). However, although intriguing, the fact that MMP-9 cleavage of collagen in vitro results in increased chemotaxis, does not confirm that this is the mechanism for MMP-9-associated increases in inflammation. Of note, in our studies of murine Lyme arthritis, we found differences in inflammation between the WT and MMP-9 −/−
mice in the ankle joints but not in the heart. Both type I and type IV collagen are located at these sites (9
); however, given the differences in tissue structure and the accessibility of the collagen to MMP-9, the resultant concentrations of the chemotactic molecule may be different. Alternatively, we may have missed a difference in inflammation in the hearts since we only looked for carditis 4 weeks postinfection. Murine Lyme-induced carditis usually peaks around 2 weeks postinfection, whereas arthritis peaks at 3 to 4 weeks (4
). By sacrificing the mice at 3.5 weeks, we would have missed any differences that are detectable only at the peak of inflammation.
An interesting point that is raised by our data is whether the recruitment of large numbers of inflammatory cells is needed to control spirochete infection. In spite of the lower amounts of macrophages and neutrophils in the infiltrate, the spirochetal burdens in the two groups of mice were remarkably similar. Other studies have shown that the converse may also be true. Heavy inflammatory cell infiltration in MyD88−/− mice was not effective in clearing bacteria and these mice showed 1- to 2-log higher bacterial burdens despite an increase in inflammatory cells. In our study, loss of MMP-9 that resulted in decreased inflammation did not hinder the ability of the animal to control infection to WT levels. Given that damage to host tissues in B. burgdorferi infection occurs largely through the action of host proteases rather than proteases produced by the bacteria, these data suggest that it may be possible to limit inflammation and cellular damage without hindering immune mediated clearance of the organism. Further studies will be required to understand the contributions of MMPs and host immune cells to cellular damage and control of B. burgdorferi infection.