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1.  Induction of Host Matrix Metalloproteinases by Borrelia burgdorferi Differs in Human and Murine Lyme Arthritis  
Infection and Immunity  2005;73(1):126-134.
Matrix metalloproteinases (MMPs) are induced from host tissues in response to Borrelia burgdorferi. Upregulation of MMPs may play a role in the dissemination of the organism through extracellular matrix tissues, but it can also result in destructive pathology. Although mice are a well-accepted model for Lyme arthritis, there are significant differences compared to human disease. We sought to determine whether MMP expression could account for some of these differences. MMP expression patterns following B. burgdorferi infection were analyzed in primary human chondrocytes, synovial fluid samples from patients with Lyme arthritis, and cartilage tissue from Lyme arthritis-susceptible and -resistant mice by using a gene array, real-time PCR, an enzyme-linked immunosorbent assay, and immunohistochemistry. B. burgdorferi infection significantly induced transcription of MMP-1, -3, -13, and -19 from primary human chondrocyte cells. Transcription of MMP-10 and tissue inhibitor of metalloprotease 1 was increased with B. burgdorferi infection, but protein expression was only minimally increased. The synovial fluid levels of MMPs from patients with high and low spirochete burdens were consistent with results seen in the in vitro studies. B. burgdorferi-susceptible C3H/HeN mice infected with B. burgdorferi showed induction of MMP-3 and MMP-19 but no other MMP or tissue inhibitor of metalloprotease. As determined by immunohistochemistry, MMP-3 expression was increased only in chondrocytes near the articular surface. The levels of MMPs were significantly lower in the more Lyme arthritis-resistant BALB/c and C57BL/6 mice. Differences between human and murine Lyme arthritis may be related to the lack of induction of collagenases, such MMP-1 and MMP-13, in mouse joints.
doi:10.1128/IAI.73.1.126-134.2005
PMCID: PMC539001  PMID: 15618147
2.  Borrelia Spirochetes Upregulate Release and Activation of Matrix Metalloproteinase Gelatinase B (MMP-9) and Collagenase 1 (MMP-1) in Human Cells 
Infection and Immunity  2001;69(1):456-462.
Borrelia burgdorferi, the spirochetal agent of Lyme disease, stimulated human peripheral blood monocytes to release pro-matrix metalloproteinase-9 (gelatinase B; pro-MMP-9) and active matrix metalloproteinase-1 (collagenase-1; MMP-1). Human neutrophils also released pro-MMP-9 and a 130-kDa protein with gelatinolytic activity in response to live B. burgdorferi. In addition, U937 cells and human keratinocyte cells were also stimulated to release pro-MMP-9 under the same conditions. However, human umbilical vein endothelial cells (HUVECs) released pro-MMP-9 and pro-MMP-2 in a constitutive manner and were not influenced by live spirochetes. MMPs produced by human monocytes also enhanced the penetration of B. burgdorferi through extracellular matrix component barriers in vitro. Plasmin stabilized on the surface of the Lyme disease spirochete was shown to activate pro-MMP-9 to its active form. This active form was also observed in the plasma of mice infected with a relapsing fever borrelia. These results suggest that borreliae can upregulate MMPs and possibly mediate an activation cascade initiated by plasmin bound to the microbial surface. MMPs may play a role in dissemination of the Lyme disease spirochete and in the pathogenesis of Borrelia infection.
doi:10.1128/IAI.69.1.456-462.2001
PMCID: PMC97903  PMID: 11119537
3.  β2 Integrins Control the Severity of Murine Lyme Carditis  
Infection and Immunity  2005;73(6):3242-3250.
Infection of C57BL/6 (B6) mice with the Lyme disease spirochete Borrelia burgdorferi can result in development of arthritis and carditis. B. burgdorferi induces expression of β2/CD18 integrins, adhesion molecules that mediate the firm adhesion of leukocytes to the endothelium necessary for cellular extravasation during inflammation. The important role of β2/CD18 integrins during extravasation suggests that these molecules play a role in the development of Lyme arthritis and carditis. The dependency of these inflammatory processes on the β2 integrins was investigated in CD18 hypomorph mice, which express low levels of CD18. The results indicate that CD18 deficiency did not abrogate development of Lyme arthritis or carditis. Moreover, it resulted in increased severity of Lyme carditis. B. burgdorferi-infected CD18 hypomorph mice showed an increased macrophage infiltration of the heart, while they produced lower levels of borreliacidal anti-B. burgdorferi antibodies compared to wild-type mice. In accordance with these results, we demonstrate that dendritic cells from CD18 hypomorph mice secrete higher levels of monocyte/macrophage chemoattractant protein 1 (MCP-1/CCL2) in response to B. burgdorferi. Similarly, we show by real-time PCR that B. burgdorferi-infected hearts from CD18 hypomorph mice express increased levels of MCP-1 RNA compared to wild-type mice. Overall, our results indicate that β2 integrin deficiency does not abrogate B. burgdorferi-induced inflammation; rather, it results in increased recruitment of macrophages into the B. burgdorferi-infected heart, likely due to the increased expression of MCP-1 in this tissue. Thus, β2 integrins may play a regulatory role in B. burgdorferi-induced inflammation beyond mediating adhesion of leukocytes to the endothelium.
doi:10.1128/IAI.73.6.3242-3250.2005
PMCID: PMC1111855  PMID: 15908348
4.  MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability 
PLoS ONE  2012;7(8):e42596.
Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13−/−) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13−/− mice. Granulation tissue in Mmp13−/− mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13−/− mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13−/− mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13−/− granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13−/− mice compared to WT mice. Mmp13−/− mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.
doi:10.1371/journal.pone.0042596
PMCID: PMC3413640  PMID: 22880047
5.  Impaired remodeling phase of fracture repair in the absence of matrix metalloproteinase-2 
Disease Models & Mechanisms  2010;4(2):203-211.
SUMMARY
The matrix metalloproteinase (MMP) family of extracellular proteases performs crucial roles in development and repair of the skeleton owing to their ability to remodel the extracellular matrix (ECM) and release bioactive molecules. Most MMP-null skeletal phenotypes that have been previously described are mild, thus permitting the assessment of their functions during bone repair in the adult. In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype. In this study, we assessed the role of MMP2 during mouse fracture repair and compared it with the roles of MMP9 and MMP13. Mmp2 was expressed at low levels in the normal skeleton and was broadly expressed in the fracture callus. Treatment of wild-type mice with a general MMP inhibitor, GM6001, caused delayed cartilage remodeling and bone formation during fracture repair, which resembles the defect observed in Mmp9–/– mice. Unlike Mmp9- and Mmp13-null mutations, which affect both cartilage and bone in the callus, the Mmp2-null mutation delayed bone remodeling but not cartilage remodeling. This remodeling defect occurred without changes in either osteoclast recruitment or vascular invasion of the fracture callus compared with wild type. However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2-null mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13-null mice. In keeping with the skeletal phenotype of Mmp2-null mice, MMP2 plays a role in the remodeling of new bone within the fracture callus and impacts later stages of bone repair compared with MMP9 and MMP13. Taken together, our results indicate that MMPs play unique and distinct roles in regulating skeletal tissue deposition and remodeling during fracture repair.
doi:10.1242/dmm.006304
PMCID: PMC3046093  PMID: 21135056
6.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
Introduction:
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
Objectives:
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Results:
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
Discussion:
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
7.  Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis 
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.
In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCδ inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCδ, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.
doi:10.1186/ar2021
PMCID: PMC1779381  PMID: 16872482
8.  Mast Cell-restricted, Tetramer-forming Tryptases Induce Aggrecanolysis in Articular Cartilage by Activating Matrix Metalloproteinase-3 and -13 Zymogens1 
Mouse mast cell protease (mMCP)-6-null C57BL/6 mice lost less aggrecan proteoglycan from the extracellular matrix of their articular cartilage during inflammatory arthritis than wild-type (WT) C57BL/6 mice, suggesting that this mast cell (MC)-specific mouse tryptase plays prominent roles in articular cartilage catabolism. We used ex vivo mouse femoral head explants to determine how mMCP-6 and its human ortholog hTryptase-β mediate aggrecanolysis. Exposure of the explants to recombinant hTryptase-β, recombinant mMCP-6, or lysates harvested from WT mouse peritoneal MCs (PMCs) significantly increased the levels of enzymatically active matrix metalloproteinases (MMP) in cartilage and significantly induced aggrecan loss into the conditioned media, relative to replicate explants exposed to medium alone or lysates collected from mMCP-6-null PMCs. Treatment of cartilage explants with tetramer-forming tryptases generated aggrecan fragments that contained C-terminal DIPEN and N-terminal FFGVG neoepitopes, consistent with MMP-dependent aggrecanolysis. In support of these data, hTryptase-β was unable to induce aggrecan release from the femoral head explants obtained from Chloe mice that resist MMP cleavage at the DIPEN↓FFGVG site in the interglobular domain of aggrecan. In addition, the abilities of mMCP-6-containing lysates from WT PMCs to induce aggrecanolysis were prevented by inhibitors of MMP-3 and MMP-13. Finally, recombinant hTryptase-β was able to activate latent pro-MMP-3 and pro-MMP-13 in vitro. The accumulated data suggest that human and mouse tetramer-forming tryptases are MMP convertases that mediate cartilage damage and the proteolytic loss of aggrecan proteoglycans in arthritis, in part, by activating the zymogen forms of MMP-3 and MMP-13 which are constitutively present in articular cartilage.
doi:10.4049/jimmunol.1300856
PMCID: PMC3727140  PMID: 23797671
mast cells; inflammation; arthritis; cartilage; aggrecan proteoglycan; matrix metalloproteinases; tryptase
9.  Dual role for Fcγ receptors in host defense and disease in Borrelia burgdorferi-infected mice 
Arthritis in mice infected with the Lyme disease spirochete, Borrelia burgdorferi, results from the influx of innate immune cells responding to the pathogen in the joint and is influenced in part by mouse genetics. Production of inflammatory cytokines by innate immune cells in vitro is largely mediated by Toll-like receptor (TLR) interaction with Borrelia lipoproteins, yet surprisingly mice deficient in TLR2 or the TLR signaling molecule MyD88 still develop arthritis comparable to that seen in wild type mice after B. burgdorferi infection. These findings suggest that other, MyD88-independent inflammatory pathways can contribute to arthritis expression. Clearance of B. burgdorferi is dependent on the production of specific antibody and phagocytosis of the organism. As Fc receptors (FcγR) are important for IgG-mediated clearance of immune complexes and opsonized particles by phagocytes, we examined the role that FcγR play in host defense and disease in B. burgdorferi-infected mice. B. burgdorferi-infected mice deficient in the Fc receptor common gamma chain (FcεRγ−/− mice) harbored ~10 fold more spirochetes than similarly infected wild type mice, and this was associated with a transient increase in arthritis severity. While the elevated pathogen burdens seen in B. burgdorferi-infected MyD88−/− mice were not affected by concomitant deficiency in FcγR, arthritis was reduced in FcεRγ−/−MyD88−/− mice in comparison to wild type or single knockout mice. Gene expression analysis from infected joints demonstrated that absence of both MyD88 and FcγR lowers mRNA levels of proteins involved in inflammation, including Cxcl1 (KC), Xcr1 (Gpr5), IL-1beta, and C reactive protein. Taken together, our results demonstrate a role for FcγR-mediated immunity in limiting pathogen burden and arthritis in mice during the acute phase of B. burgdorferi infection, and further suggest that this pathway contributes to the arthritis that develops in B. burgdorferi-infected MyD88−/− mice.
doi:10.3389/fcimb.2014.00075
PMCID: PMC4052197  PMID: 24967215
Fc receptor; Borrelia burgdorferi; arthritis; mice; Lyme disease; toll-like receptors; MyD88
10.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
Introduction:
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
Aims:
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Methods:
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
Results:
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Discussion:
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
11.  Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis 
Arthritis Research & Therapy  2013;15(6):R222.
Introduction
Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.
Methods
For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.
Results
This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.
Conclusions
MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.
doi:10.1186/ar4423
PMCID: PMC3979078  PMID: 24369907
12.  Adenoviral Delivery of Interleukin-10 Fails To Attenuate Experimental Lyme Disease▿  
Infection and Immunity  2008;76(12):5500-5507.
Production of interleukin-10 (IL-10) by C57BL/6 mice following infection with Borrelia burgdorferi has been proposed as a mechanism whereby resistance to the development of experimental Lyme arthritis is maintained. In the current study, we sought to determine the role of IL-10 during infection of arthritis- and carditis-susceptible C3H mice. Infection of C3H IL-10−/− mice led to increased joint swelling and arthritis severity scores over those of wild-type C3H mice. Measurement of B. burgdorferi numbers in joints or disseminated tissues indicated a more efficient clearance of spirochetes in the absence of IL-10, similar to that reported in C57BL/6 IL-10−/− mice. However, in contrast to previous in vitro work, infection of C3H IL-10−/− mice led to decreased in vivo expression of the cytokines KC, IL-1β, IL-4, and IL-12p70 in the infected joints. Finally, adenoviral expression of IL-10 in the infected joints of C3H mice was unable to modulate the development of severe Lyme arthritis and had no effect on spirochete clearance or Borrelia-specific antibody production. Development of Lyme carditis appeared to be independent of modulation by IL-10. These results suggest that IL-10 limits the development of joint inflammation in both arthritis-resistant and -susceptible mouse strains infected with B. burgdorferi and that increased IL-10 production cannot rescue genetic susceptibility to development of pathology in this model.
doi:10.1128/IAI.00808-08
PMCID: PMC2583579  PMID: 18824530
13.  Biochemical Characterization and N-terminomics Analysis of Leukolysin, the Membrane-type 6 Matrix Metalloprotease (MMP25) 
The Journal of Biological Chemistry  2012;287(16):13382-13395.
Background: Neutrophil-specific membrane-type 6 matrix metalloproteinase (MT6-MMP)/leukolysin has seven known substrates.
Results: We identified 72 new MT6-MMP substrates by proteomics and family-wide chemokine screens. Cell membrane-bound vimentin chemoattracts macrophages, whereas MT6-MMP-cleaved vimentin is an “eat-me” signal greatly increasing phagocytosis.
Conclusion: MT6-MMP substrates indicate a role for clearance of apoptotic neutrophils.
Significance: MT6-MMP cleaves many bioactive proteins important in innate immunity.
The neutrophil-specific protease membrane-type 6 matrix metalloproteinase (MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet remains poorly characterized. To characterize the biological roles of MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized MT6-MMP, profiled its tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26 chemokines for cleavage using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6′ from which an unusual glutamate preference in P1 was identified. The degradomics screen terminal amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular “moonlighting” form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin potently stimulated phagocytosis, which was not a property of the full-length protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating “eat-me” signals upon vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve inflammation.
doi:10.1074/jbc.M111.314179
PMCID: PMC3339980  PMID: 22367194
Chemokines; Inflammation; Matrix Metalloproteinase (MMP); Phagocytosis; Proteolytic Enzymes; Proteomics
14.  Borrelia burgdorferi-Induced Expression of Matrix Metalloproteinases from Human Chondrocytes Requires Mitogen-Activated Protein Kinase and Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathways  
Infection and Immunity  2004;72(5):2864-2871.
Elevations in matrix metalloproteinase 1 (MMP-1) and MMP-3 have been found in patients with Lyme arthritis and in in vitro models of Lyme arthritis using cartilage explants and chondrocytes. The pathways by which B. burgdorferi, the causative agent of Lyme disease, induces the production of MMP-1 and MMP-3 have not been elucidated. We examined the role of the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in MMP induction by B. burgdorferi. Infection with B. burgdorferi results in rapid phosphorylation of p38 and JNK within 15 to 30 min. Inhibition of JNK and p38 MAPK significantly reduced B. burgdorferi-induced MMP-1 and MMP-3 expression. Inhibition of ERK1/2 completely inhibited the expression of MMP-3 in human chondrocytes following B. burgdorferi infection but had little effect on the expression of MMP-1. B. burgdorferi infection also induced phosphorylation and nuclear translocation of STAT-3 and STAT-6 in primary human chondrocytes. Expression of MMP-1 and MMP-3 was significantly inhibited by inhibition of JAK3 activity. Induction of MMP-1 and -3 following MAPK and JAK/STAT activation was cycloheximide sensitive, suggesting synthesis of intermediary proteins is required. Inhibition of tumor necrosis factor alpha (TNF-α) significantly reduced MMP-1 but not MMP-3 expression from B. burgdorferi-infected cells; inhibition of interleukin-1β (IL-1β) had no effect. Treatment of B. burgdorferi-infected cells with JAK and MAPK inhibitors significantly inhibited TNF-α induction, consistent with at least a partial role for TNF-α in B. burgdorferi-induced MMP-1 expression in chondrocytes.
doi:10.1128/IAI.72.5.2864-2871.2004
PMCID: PMC387916  PMID: 15102798
15.  Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host 
PLoS Pathogens  2008;4(6):e1000090.
Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.
Author Summary
Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme disease. They exhibit an unusual form of motility and can infect many different tissues; however, the mechanism by which they disseminate from the blood to target sites is unknown. Direct visualization of bacterial pathogens at the single cell level in living hosts is an important goal of microbiology, since this approach is likely to yield critical insight into disease processes. We engineered a fluorescent strain of Borrelia burgdorferi, a Lyme disease pathogen, and used conventional and spinning disk confocal intravital microscopy to directly visualize these bacteria in real time and 3D in living mice. We found that spirochete interaction with and dissemination out of the vasculature was a multi-stage process of unexpected complexity and that spirochete movement appeared to play an integral role in dissemination. To our knowledge, this is the first report of high resolution 3D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.
doi:10.1371/journal.ppat.1000090
PMCID: PMC2408724  PMID: 18566656
16.  Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3 
Arthritis Research & Therapy  2012;14(6):R247.
Introduction
The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1β, resulting in bioactive IL-1β. IL-1β is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1β in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis.
Methods
Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences.
Results
We demonstrate that ASC/caspase-1-driven IL-1β is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial.
Conclusions
Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.
doi:10.1186/ar4090
PMCID: PMC3674595  PMID: 23148704
17.  Borrelia burgdorferi basic membrane proteins A and B participate in the genesis of Lyme arthritis 
Lyme arthritis results from colonization of joints by Borrelia burgdorferi and the ensuing host response. Using gene array–based differential analysis of B. burgdorferi gene expression and quantitative reverse trancription-polymerase chain reaction, we identified two paralogous spirochete genes, bmpA and bmpB, that are preferentially up-regulated in mouse joints compared with other organs. Transfer of affinity-purified antibodies against either BmpA or BmpB into B. burgdorferi–infected mice selectively reduced spirochete numbers and inflammation in the joints. B. burgdorferi lacking bmpA/B were therefore generated to further explore the role of these proteins in the pathogenesis of Lyme disease. B. burgdorferi lacking bmpA/B were infectious in mice, but unable to persist in the joints, and they failed to induce severe arthritis. Complementation of the mutant spirochetes with a wild-type copy of the bmpA and bmpB genes partially restored the original phenotype. These data delineate a role for differentially produced B. burgdorferi antigens in spirochete colonization of mouse joints, and suggest new strategies for the treatment of Lyme arthritis.
doi:10.1084/jem.20070962
PMCID: PMC2234379  PMID: 18166585
18.  Discovery of a Siderophore Export System Essential for Virulence of Mycobacterium tuberculosis 
PLoS Pathogens  2013;9(1):e1003120.
Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. Mycobacterium tuberculosis (Mtb) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an Mtb mutant lacking the mmpS4 and mmpS5 genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of mmpS4 and mmpS5 did not change uptake of carboxymycobactin by Mtb. Thin layer chromatography showed that the ΔmmpS4/S5 mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in Mtb and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the mmpS4/S5 deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type Mtb. This is the strongest attenuation observed so far for Mtb mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of Mtb.
Author Summary
In the late 19th century the French physician Armand Trousseau recognized that treating anemic tuberculosis patients with iron salts exacerbated the disease. In 1911 Twort postulated that mycobacteria produce an essential growth factor which was identified in 1953 as mycobactin. The hydrophobic mycobactin and its more water-soluble cousin carboxymycobactin are small molecules made by Mycobacterium tuberculosis to scavenge iron from its human host. While the biosynthesis of these siderophores has been decoded, it was unknown how M. tuberculosis secretes these molecules. In this study, we identified two similar transport systems, MmpS4/MmpL4 and MmpS5/MmpL5, which are required for biosynthesis and export of siderophores by M. tuberculosis. The lack of these transport systems drastically decreased the number of M. tuberculosis cells in the lungs and spleens of infected mice. Lung examination and histological assessment in mice infected with the mmpS4/S5 deletion strain showed almost no signs of infection. Further, none of the mice infected with this strain died within 180 days in contrast to wild-type M. tuberculosis. In this study, we identified the first components of a novel siderophore export system in M. tuberculosis and showed the importance of siderophore export for virulence of M. tuberculosis.
doi:10.1371/journal.ppat.1003120
PMCID: PMC3561183  PMID: 23431276
19.  Matrix Metalloproteinases Promote Inflammation and Fibrosis in Asbestos-Induced Lung Injury in Mice 
Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7–28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.
doi:10.1165/rcmb.2005-0471OC
PMCID: PMC1820635  PMID: 16574944
asbestos; extracellular superoxide dismutase; matrix metalloproteinase; pulmonary fibrosis
20.  Matrix Metalloproteinases Promote Inflammation and Fibrosis in Asbestos-Induced Lung Injury in Mice 
Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7–28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.
doi:10.1165/rcmb.2005-0471OC
PMCID: PMC1820635  PMID: 16574944
asbestos; extracellular superoxide dismutase; matrix metalloproteinase; pulmonary fibrosis
21.  The Effect of MMP2 and MMP 2/9 deletion in Experimental Post-thrombotic Vein Wall Remodeling 
Journal of vascular surgery  2013;58(5):10.1016/j.jvs.2012.11.088.
Introduction
Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP2 contributes to vein wall remodeling after VT is unknown.
Methods
Stasis VT was produced by ligation of the inferior vena cava (IVC) and tissue was harvested at 2, 8, and 21 days in MMP2 −/− and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, ELISA, real time PCR, and zymography was performed.
Results
Thrombus resolution was impaired at 8d in MMP2 −/− as compared with WT, evidenced by a 51% increase in VT size (p < .01), and 3 fold fewer vWF positive channels (p<.05). In MMP2 −/− mice, the main phenotypic fibrotic differences occurred at 8d post VT, with significantly less vein wall collagen content (p=.013), 4 fold lower procollagen III gene expression (p < .01) but no difference in procollagen I as compared to WT. Decreased inflammation in MMP2−/− vein walls was suggested by ~ 3 fold reduced TNFα and IL1β at 2d and 8d post VT (p < .05). A 4 fold increase in vein wall monocytes (p = .03) with 3 fold decreased apoptosis (p < .05), but no difference in cellular proliferation at 8d was found in MMP2−/− as compared with WT. As increased compensatory MMP9 activity was observed in the MMP2 −/− mice, MMP2/9 double null mice had thrombus induced with VT harvest at 8d. Consistently, 2 fold larger VT, a 3 fold decrease in vein wall collagen, and a 3 fold increase in monocytes was found (all p < .05). Similar findings were observed in MMP9 −/− mice administered an exogenous MMP2 inhibitor.
Conclusion
In stasis VT, deletion of MMP2 was associated with less midterm vein wall fibrosis and inflammation, despite an increase in monocytes. Consideration that VT resolution was impaired with MMP2 (and MMP2/9) deletion suggests direct inhibition will likely also require anticoagulant therapy.
doi:10.1016/j.jvs.2012.11.088
PMCID: PMC3688659  PMID: 23490298
Venous thrombosis; inflammation; matrix metalloproteinases; monocytes
22.  Overlapping and enzyme-specific contributions of matrix metalloproteinases-9 and -12 in IL-13–induced inflammation and remodeling 
IL-13 potently stimulates eosinophilic and lymphocytic inflammation and alveolar remodeling in the lung, effects that depend on the induction of various matrix metalloproteinases (MMPs). Here, we compared the remodeling and inflammatory effects of an IL-13 transgene in lungs of wild-type, MMP-9–deficient, or MMP-12–deficient mice. IL-13–induced alveolar enlargement, lung enlargement, compliance alterations, and respiratory failure and death were markedly decreased in the absence of MMP-9 or MMP-12. Moreover, IL-13 potently induced MMPs-2, -12, -13, and -14 in the absence of MMP-9, while induction of MMPs-2, -9, -13, and -14 by IL-13 was diminished in the absence of MMP-12. A deficiency in MMP-9 did not alter eosinophil, macrophage, or lymphocyte recovery, but increased the recovery of total leukocytes and neutrophils in bronchoalveolar lavage (BAL) fluids from IL-13 transgenic mice. In contrast, a deficiency in MMP-12 decreased the recovery of leukocytes, eosinophils, and macrophages, but not lymphocytes or neutrophils. These studies demonstrate that IL-13 acts via MMPs-9 and -12 to induce alveolar remodeling, respiratory failure, and death and that IL-13 induction of MMPs-2, -9, -13, and -14 is mediated at least partially by an MMP-12–dependent pathway. The also demonstrate that MMPs-9 and -12 play different roles in the generation of IL-13–induced inflammation, with MMP-9 inhibiting neutrophil accumulation and MMP-12 contributing to the accumulation of eosinophils and macrophages.
doi:10.1172/JCI14136
PMCID: PMC150413  PMID: 12189240
23.  Strain-Specific Variation of the Decorin-Binding Adhesin DbpA Influences the Tissue Tropism of the Lyme Disease Spirochete 
PLoS Pathogens  2014;10(7):e1004238.
Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.
Author Summary
Lyme disease, the most common vector-borne disease in the United States, is caused by a bacterium, Borrelia burgdorferi. This bacterium infects the skin at the site of the tick bite and then can spread to other tissues, such as the heart, joints or nervous system, causing carditis, arthritis or neurologic disease. To colonize human tissues, the pathogen produces surface proteins that promote bacterial attachment to these sites. For example, DbpA binds to decorin, a component of human tissue. Different Lyme disease strains differ in the particular tissues they colonize and the disease they cause, but we do not understand why. Different strains also make distinct versions of DbpA that bind decorin differently, so variation of DbpA might contribute to strain-to-strain variation in clinical manifestations. To test this, we infected mice with Lyme disease strains that were identical except for the particular DbpA variant they produced. We found that the strains colonized different tissues and caused different diseases, such as arthritis or carditis. These results provide the first solid evidence that variation of an outer surface protein, in this case DbpA, influences what tissues are most affected during Lyme disease.
doi:10.1371/journal.ppat.1004238
PMCID: PMC4117581  PMID: 25079227
24.  Recruitment of Macrophages and Polymorphonuclear Leukocytes in Lyme Carditis▿  
Infection and Immunity  2006;75(2):613-620.
Lyme arthritis, caused by the spirochete Borrelia burgdorferi, can be recurrent or prolonged, whereas Lyme carditis is mostly nonrecurring. A prominent difference between arthritis and carditis is the differential representation of phagocytes in these lesions: polymorphonuclear leukocytes (PMN) are more prevalent in the joint, and macrophages predominate in the heart lesion. We have previously shown differential efficiency of B. burgdorferi clearance by PMN and macrophages, and we now investigate whether these functional differences at the cellular level may contribute to the observed differences in organ-specific pathogenesis. When we infected mice lacking the neutrophil chemokine receptor (CXCR2−/− mice) with spirochetes, we detected fewer PMN in joints and less-severe arthritis. Here we have investigated the effects of the absence of the macrophage chemokine receptor CCR2 on the development and resolution of Lyme carditis in resistant (C57BL/6J [B6]) and sensitive (C3H/HeJ [C3H]) strains of mice. In B6 CCR2−/− mice, although inflammation in hearts is mild, we detected an increased burden of B. burgdorferi compared to that in wild-type (WT) mice, suggesting reduced clearance in the absence of macrophages. In contrast, C3H CCR2−/− mice have severe inflammation but a decreased B. burgdorferi burden compared to that in WT C3H mice both at peak disease and during resolution. Histopathologic examination of infected hearts revealed that infected C3H CCR2−/− animals have an increased presence of PMN, suggesting compensatory mechanisms of B. burgdorferi clearance in the hearts of infected C3H CCR2−/− mice. The more efficient clearance of B. burgdorferi from hearts by CCR2−/− versus WT C3H mice suggests a natural defect in the recruitment or function of macrophages in C3H mice, which may contribute to the sensitivity of this strain to B. burgdorferi infection.
doi:10.1128/IAI.00685-06
PMCID: PMC1828503  PMID: 17101663
25.  Matrix Metalloprotease 1a-Deficiency Suppresses Tumor Growth and Angiogenesis 
Oncogene  2013;33(17):2264-2272.
Matrix metalloprotease-1 (MMP1) is an important mediator of tumorigenesis, inflammation and tissue remodeling through its ability to degrade critical matrix components. Recent studies indicate that stromal-derived MMP1 may exert direct oncogenic activity by signaling through protease-activated receptor-1 (PAR1) in carcinoma cells, however, this has not been established in vivo. We generated a Mmp1a knock-out mouse to ascertain whether stromal-derived Mmp1a affects tumor growth. Mmp1a-deficient mice are grossly normal and born in Mendelian ratios, however, deficiency of Mmp1a results in significantly decreased growth and angiogenesis of lung tumors. Co-implantation of lung cancer cells with wild-type Mmp1a+/+ fibroblasts completely restored tumor growth in Mmp1a-deficient animals, highlighting the critical role of stromal-derived Mmp1a. Silencing of PAR1 expression in the lung carcinoma cells phenocopied stromal Mmp1a-deficiency, thus validating tumor-derived PAR1 as a Mmp1a target. Mmp1a secretion is controlled by the ability of its prodomain to suppress auto-cleavage, whereas human MMP1 is efficiently secreted due to stable pro- and catalytic domain interactions. Together, these data demonstrate that stromal Mmp1a drives in vivo tumorigenesis and provide proof-of-concept that targeting the MMP1-PAR1 axis may afford effective treatments of lung cancer.
doi:10.1038/onc.2013.157
PMCID: PMC4221191  PMID: 23708660
angiogenesis; lung cancer; Mmp1a; MMP1; PAR1

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