Experimental and Theoretical Investigation of Multispecies Oral Biofilm Resistance to Chlorhexidine Treatment.

We investigate recovery of multispecies oral biofilms following chlorhexidine gluconate (CHX) and CHX with surface modifiers (CHX-Plus) treatment. Specifically, we examine the percentage of viable bacteria in the biofilms following their exposure to CHX and CHX-Plus for 1, 3, and 10 minutes, respectively. Before antimicrobial treatment, the biofilms are allowed to grow for three weeks. We find that (a). CHX-Plus kills bacteria in biofilms more effectively than the regular 2% CHX does, (b). cell continues to be killed for up to one week after exposure to the CHX solutions, (c). the biofilms start to recover after two weeks, the percentage of the viable bacteria recovers in the 1 and 3 minutes treatment groups but not in the 10 minutes treatment group after five weeks, and the biofilms fully return to the pretreatment levels after eight weeks. To understand the mechanism, a mathematical model for multiple bacterial phenotypes is developed, adopting the notion that bacterial persisters exist in the biofilms together with regulatory quorum sensing molecules and growth factor proteins. The model reveals the crucial role played by the persisters, quorum sensing molecules, and growth factors in biofilm recovery, accurately predicting the viable bacterial population after CHX treatment.

Macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis.

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12(-/-) mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.

Matrix metalloproteinase 8 deficiency in mice exacerbates inflammatory arthritis through delayed neutrophil apoptosis and reduced caspase 11 expression.

OBJECTIVE: Neutrophil accumulation is balanced by both cell infiltration and cell clearance, the controls of which are pivotal in the pathogenesis of rheumatoid arthritis (RA) and other chronic inflammatory diseases. Of the neutrophil-specific proteases, matrix metalloproteinase 8 (MMP-8; also known as neutrophil collagenase or collagenase 2) is traditionally viewed as being crucial for collagen degradation and hence cell migration and infiltration. This study was undertaken to examine the role of MMP-8 in a murine model of spontaneous RA. METHODS: MMP-8(-/-) mice were backcrossed onto the Fas-defective MRL/lpr background, a mouse strain characterized by systemic autoimmunity including spontaneous autoimmune arthritis. Arthritis was induced with Freund's complete adjuvant and clinical disease and histologic parameters were assessed. RESULTS: MMP-8(-/-) mice had earlier and more severe joint inflammation than their MMP-8(+/+) counterparts, coupled with a massive accumulation of neutrophils in synovial tissue, an unexpected result considering the commonly held view that MMP-8 has important extracellular matrix-degradative functions. Protease and protease inhibitor analysis of MMP-8(-/-) mouse neutrophils by CLIP-CHIP microarray revealed very little additional change in protease levels except for low expression of the apoptosis initiator caspase 11. This was confirmed at the protein level in unstimulated, lipopolysaccharide-treated, and interferon-γ-treated MMP-8(-/-) mouse neutrophils. Downstream of caspase 11, the activity of the apoptosis executioner caspase 3 was consequently reduced in MMP-8(-/-) mouse neutrophils, translating to reduced neutrophil apoptosis and cell accumulation compared with wild-type mouse cells. CONCLUSION: Our findings indicate that MMP-8 is not essential for neutrophil migration in arthritis and likely other autoimmune diseases. Rather, MMP-8 is important for normal rates of neutrophil apoptosis and hence regulates cell clearance. Because MMP-8 deficiency leads to an exaggerated accumulation of neutrophil infiltrates due to delayed apoptosis and concurrent pathologic changes associated with dramatically increased neutrophil infiltration, MMP-8 is antiinflammatory and therefore a drug antitarget in the treatment of arthritis.

Matrix metalloproteinase processing of CXCL11/I-TAC results in loss of chemoattractant activity and altered glycosaminoglycan binding.

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.

Aggrecan components differentially modulate nerve growth factor-responsive and neurotrophin-3-responsive dorsal root ganglion neurite growth.

Aggrecan is one of the major chondroitin sulfate proteoglycans (CSPGs) expressed in the central nervous system. The signaling pathways activated downstream of cell interaction with aggrecan and with CSPGs in general and the importance of chondroitin sulfate-glycosaminoglycan side chains in their inhibition are unclear. Therefore, to analyze the effect of different components of aggrecan in inhibiting neurite growth, neurite outgrowth was quantified in an in vitro model in which chick dorsal root ganglion (DRG) explants were grown on substrates containing aggrecan bound to hyaluronan and link protein as a macromolecular aggregate, aggrecan monomers, hyaluronan, or ChABC-treated aggrecan. Aggrecan aggregate, aggrecan monomer, and hyaluronan inhibited neurite outgrowth from nerve growth factor (NGF)- and neurotrophin-3 (NT3)-responsive DRG neurons. Aggrecan inhibition was dependent on its chondroitin sulfate-glycosaminoglycans, as ChABC digestion alleviated neurite inhibition because of aggrecan. Growth cones displayed full or partial collapse on aggrecan aggregate, hyaluronan, and ChABC-treated aggrecan. Inhibition of Rho kinase (ROCK) with Y27632 increased neurite growth on some but not all of the aggrecan components tested. With NGF in the culture medium, Y27632 increased neurite outgrowth on aggrecan aggregate, monomers, and ChABC-treated aggrecan, but not on hyaluronan. The ROCK inhibitor also increased NT3-responsive outgrowth on aggrecan aggregate and hyaluronan, but not on ChABC-treated aggrecan. This study showed that the matrix proteoglycan aggrecan and its components have multiple effects on neurite outgrowth and that some of these effects involve the Rho/ROCK pathway.