Co-opting Host Proteases in the Pathogenesis of Lyme Arthritis
Lyme arthritis differs from other causes of bacterial septic arthritis in both tempo and outcome. In septic arthritis resulting from infection by bacteria such as Staphylococcus aureus and β-hemolytic streptococci, a combination of bacterial proteases and host proteases from inflammatory cells has been shown to lead to rapid destruction of articular cartilage. In contrast, Lyme arthritis is a slowly progressing process and synovial fluid studies typically reveal only moderate elevations in inflammatory cells. We have found that Borrelia burgdorferi does not secrete any enzymes capable of digesting collagen, laminin or gelatin and a search of its genome does not reveal any open reading frames with significant identity to exported bacterial proteases capable of digesting these proteins. Instead, it appears that Lyme arthritis may be more similar to non-infectious arthritides such as rheumatoid or osteoarthritis, where a family of host enzymes called matrix metalloproteinases (MMPs) have been implicated. We have found that at least two MMPs, MMP-1 and MMP-3, are elevated in the synovial fluid of patients with Lyme arthritis. In vitro models of Lyme arthritis using primary human chondrocytes cells have confirmed the ability of B. burgdorferi to induce MMP production and of MMP inhibitors to block cartilage degradation.
Figure 1. Localization of MMP-3 expression in vivo in murine joints. Expression of MMP-3 was determined in tissues from mice infected with B. burgdorferi by immunohistochemical staining. Boxed areas of brown staining in B. burgdorferi infected mice (bottom) indicate the presence of MMP-3. Similar areas in uninfected mice (top) are also boxed to show absence of stain for MMP-3. Red arrows point to examples of chondrocytes. Insets are the magnified views of the boxed areas.
The ability to induce MMPs may play a role in a number of different aspects of Lyme disease pathogenesis including the ability of the organism to spread through extracellular matrix, generation of host components that may be involved in environmental adaptation by the organism and modulation of the host immune response through cleavage of cytokines, chemokines and cell surface receptors. Differences in the MMPs induced may be responsible for differences in the clinical progression of the disease in patients with untreated and treated Lyme arthritis and may also be responsible for differences in arthritic manifestations of Lyme disease between humans and animal models.
The laboratory is currently focused on investigating the early events in host response to B. burgdorferi in various host tissues to better understand the signaling pathways involved in MMP induction. We are also interested in examining the potential role of MMPs in modulating the immune response to B. burgdorferi and allowing development of chronic Lyme borreliosis.
Mechanisms of Host Adaptation by B. burgdorferi
The Borrelia burgdorferi infectious cycle requires the organism to survive in both tick and mammalian hosts. Previous studies have shown that B. burgdorferi accomplishes the adjustment to the vast differences in environmental challenges presented by these hosts in part by regulating expression of its surface proteins. We have identified a host molecule, the adrenergic hormone norepinephrine (NE), which is recognized by B. burgdorferi as a signal for a specific environmental change. NE binds specifically to B. burgdorferi. The presence of NE increases production of outer surface protein A (OspA) of B. burgdorferi, a protein critical in tick colonization. Up-regulation of OspA in the presence of NE can be blocked by the use of competitive inhibitors such as propranolol.
We have also been able to show that propranolol inhibits OspA expression in an in vivo model. B. burgdorferi from ticks that were fed on infected mice treated with propranolol expressed less OspA than those fed on mice administered sham. In addition, the ticks fed on propranolol treated mice acquired B. burgdorferi at significantly lower rates than those fed on mice treated with sham suggesting that recognition of NE plays an important role in the infectious cycle of B. burgdorferi. We hypothesize that the organism may co-opt host adrenergic signals to inform of local changes that predict the presence of a tick host and allow it to prepare for transition to a new environment. We are currently working to identify genes that are co-regulated with OspA in response to NE to obtain a broader understanding of other genes that are important in adaptation to a tick host. We are also attempting to identify the B. burgdorferi receptor for catecholamines. A better understanding of how B. burgdorferi has learned to intercept host hormonal cell to cell communication and utilize it for its own purposes could lead to new strategies for controlling infection in ticks which would have a direct impact on disease transmission to humans.
Understanding Host Inflammatory Responses to B. burgdorferi
Arthritis is one of the characteristic clinical manifestations of human Lyme disease. We have previously identified signaling pathways and receptors involved in the recognition of B. burgdorferi and activation of the host immune response. Interestingly, the most widely studied receptor for borrelial products, toll-like receptor 2 (TLR2) is not strictly required for the development of arthritis and release of cytokines and chemokines. We identified a previously unrecognized receptor of B. burgdorferi products, integrin α3β1, and showed that it plays a major role in induction of inflammatory mediators. Current work focuses on understanding the contribution of the innate immune signaling pathways activated by these two receptors, integrin α3β1, and TLRs, and determining their contributions to the development of Lyme arthritis and control of infection. Integrin α3β1 appears to have both TLR2 dependent and independent mechanisms of mediating inflammatory responses to B. burgdorferi. The effects of integrin α3β1 on TLR2 dependent signaling may be in part mediated by its role in attachment and phagocytosis. Another major interest in the laboratory is in determining the mechanisms of phagocytosis of B. burgdorferi and the requirement for phagocytosis in inflammation and in control of infection. Animal studies in other organisms have suggested that integrin signaling may play a
Figure 2. MyD88 utilizes PI3K in phagocytosis of B. burgdorferi. B. burgdorferi were incubated with macrophages (wild type or MyD88-/-) and fixed and stained with an anti-borrelial antibody (red) and an anti-Arp3 antibody (green). Loss of MyD88 or inhibition of PI3K both result in loss of phagocytosis due to failure to recruit Arp3 .
major role in inflammation with only a minor impact on control of infection whereas TLR signaling is the opposite, with a major role in control of infection and a lesser role in inflammation. Because pathology in Lyme arthritis (and many other forms of arthritis) is predominantly due to activation of the host immune system, by better understanding the pathways that direct inflammation and that control infection, we hope to identify areas of divergence that will be targets for future development of therapies that can reduce the pathogenesis of disease without delaying recovery from infection.
Reduction of Vector and Reservoir Competence for Carriage of B. burgdorferi
The incidence and geographic distribution of Lyme disease in the U.S. has increased steadily since its first description in 1977. Efforts to stem the spread of the disease through controlling the population of its tick vector and/or the mouse reservoirs of the disease have met with only limited success. The only approved human vaccine to protect against Lyme disease was recently removed from the market by its manufacturer further highlighting the need for new approaches to controlling the disease. In this The incidence and geographic distribution of Lyme disease in the U.S. has increased steadily since its first description in 1977. Efforts to stem the spread of the disease through controlling the population of its tick vector and/or the mouse reservoirs of the disease have met with only limited success. The only approved human vaccine to protect against Lyme disease was recently removed from the market by its manufacturer further highlighting the need for new approaches to controlling the disease. In this project, we are developing an orally-available vaccine targeted towards the mouse and tick reservoirs of the disease. Prior attempts to vaccinate wild animals have been hampered by the lack of an efficient, oral delivery system which is both stable under natural environmental conditions and can generate an intense immune response. The photo (courtesy of Dr. Sam Telford, Cummings School of Veterinary Medicine) illustrates a mouse next box for distribution of baits.We are studying the use of a variety of viral vectors as a delivery mechanism for a targeted mouse vaccine. In addition, we are working to develop new vaccines that combine “traditional” approaches that target the pathogen with novel approaches that target the tick vectors. Preliminary data has shown that uptake of antibodies directed at tick proteins by ticks during a blood meal can inhibit feeding, prevent transmission of pathogens and even result in tick mortality. A tick directed vaccine may be particularly relevant for tick-transmitted pathogens such as Babesia microti and Anaplasma phagocytophilum where there are no advanced vaccine targets.
Figure 3. Peromyscus mouse eating bait containing the vaccinia virus based B. burgdorferi vaccine.
Bacterial Interactions in the Pathogenesis of Periodontitis
Periodontitis is the leading cause of tooth loss in the U.S. It is thought to be mediated by the interaction of bacteria with host cells leading to the production of destructive proteases. While many different bacteria have been isolated from the tissue of patients with periodontitis, no single bacterial species has emerged as essential to the development of periodontitis and it remains unclear how different bacteria contribute to the pathogenesis of the disease. The major proteases found to be involved in periodontitis come from a class of enzymes called matrix metalloproteinases (MMPs). MMPs all share a function in degradation of extracellular matrix (ECM) proteins. Multiple MMPs have been found to be elevated in the ginigval space of patients with periodontitis and therapy with MMP inhibitors has been shown to improve outcomes in patients with periodontitis. Individual bacteria have been shown to induce MMPs using in vitro systems and other bacteria have been shown to produce enyzmes which can activate MMPs or inactivate their inhibitors. However, while the role of bacterially induced MMPs in the pathogenesis of periodontitis has become clearer, the mechanisms by which MMP induction may benefit the bacteria remain unknown. Recently, it has been shown that MMPs have substrates beyond ECM proteins and may be involved in immunomodulation through cleavage of cell surface receptors or inactivation of inflammatory cytokines and chemokines. We are in the process of systematically examining the association of individual and combinations of bacterial species with pathologic increases in MMPs. With these studies, we are testing the hypothesis that different microbial communities causing periodontitis share in common the ability to induce and activate MMPs which in turn protects the community by modulating the host immune response through cleavage of chemotactic chemokines.