Macrophage MMP10 Regulates TLR7-Mediated Tolerance.

Using an in vivo model of tolerance to TLR7-induced skin inflammation, we found a critical role for macrophage-derived MMP10 in mediating immune hypo-responsiveness. Cutaneous exposure to Imiquimod (IMQ), a TLR7 agonist, induced acute expression of pro-inflammatory factors (IL1ß, IL6, CXCL1) and neutrophil influx equally in both wildtype and Mmp10-/- mice. However, whereas subsequent exposure (11 and 12 days later) to IMQ led to marked abrogation of pro-inflammatory factor expression in wildtype mice, Mmp10-/- mice responded similarly as they did to the first application. In addition, the second exposure led to increased expression of negative regulators of TLR signaling (TNFAIP3, IRAK3) and immunosuppressive cytokines (IL10, TGFß1) in wildtype mice but not in Mmp10-/- mice. In vitro studies demonstrated that prior exposure of IMQ to bone marrow-derived macrophages (BMDM) made wildtype cells refractory to subsequent stimulation but did not for Mmp10-/- macrophages. These findings expand the critical roles MMP10 plays in controlling macrophage activation to indicate that the development of immune tolerance to TLR7 ligand is dependent on this macrophage-derived proteinase.

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

Stromelysin-2 (MMP10) Moderates Inflammation by Controlling Macrophage Activation.

Several members of the matrix metalloproteinase (MMP) family control a range of immune processes, such as leukocyte influx and chemokine activity. Stromelysin-2 (MMP10) is expressed by macrophages in numerous tissues after injury; however, little is known of its function. In this study, we report that MMP10 is expressed by macrophages in human lungs from patients with cystic fibrosis and induced in mouse macrophages in response to Pseudomonas aeruginosa infection both in vivo and by isolated resident alveolar and bone marrow-derived macrophages (BMDM). Our data indicates that macrophage MMP10 serves a beneficial function in response to acute infection. Whereas wild-type mice survived infection with minimal morbidity, 50% of Mmp10(-/-) mice died and all showed sustained weight loss (morbidity). Although bacterial clearance and neutrophil influx did not differ between genotypes, macrophage numbers were ∼3-fold greater in infected Mmp10(-/-) lungs than in wild-types. Adoptive transfer of wild-type BMDM normalized infection-induced morbidity in Mmp10(-/-) recipients to wild-type levels, demonstrating that the protective effect of MMP10 was due to its production by macrophages. Both in vivo and in cultured alveolar macrophages and BMDM, expression of several M1 macrophage markers was elevated, whereas M2 markers were reduced in Mmp10(-/-) tissue and cells. Global gene expression analysis revealed that infection-mediated transcriptional changes persisted in Mmp10(-/-) BMDM long after they were downregulated in wild-type cells. These results indicate that MMP10 serves a beneficial role in response to acute infection by moderating the proinflammatory response of resident and infiltrating macrophages.

MMP-10 Regulates Collagenolytic Activity of Alternatively Activated Resident Macrophages.

Matrix metalloproteinase-10 (MMP-10) is expressed by macrophages and epithelium in response to injury, but its functions in wound repair are unknown. We observed increased collagen deposition and skin stiffness in Mmp10(-/-) wounds, with no difference in collagen expression or reepithelialization. Increased collagen deposition in Mmp10(-/-) wounds was accompanied by less collagenolytic activity and reduced expression of specific metallocollagenases, particularly MMP-8 and MMP-13, where MMP-13 was the key collagenase. Ablation and adoptive transfer approaches and cell-based models demonstrated that the MMP-10-dependent collagenolytic activity was a product of alternatively activated (M2) resident macrophages. These data demonstrate a critical role for macrophage MMP-10 in controlling the tissue remodeling activity of macrophages and moderating scar formation during wound repair.

Matrix remodeling by MMPs during wound repair.

Repair following injury involves a range of processes - such as re-epithelialization, scar formation, angiogenesis, inflammation, and more - that function, often together, to restore tissue architecture. MMPs carry out diverse roles in all of these activities. In this article, we discuss how specific MMPs act on ECM during two critical repair processes: re-epithelialization and resolution of scar tissue. For wound closure, we discuss how two MMPs - MMP1 in human epidermis and MMP7 in mucosal epithelia - facilitate re-epithelialization by cleaving different ECM or ECM-associated proteins to affect similar integrin:matrix adhesion. In scars and fibrotic tissues, we discuss that a variety of MMPs carry out a diverse range of activities that can either promote or limit ECM deposition. However, few of these MMP-driven activities have been demonstrated to be due a direct action on ECM.

uPARAP function in cutaneous wound repair.

Optimal skin wound healing relies on tight balance between collagen synthesis and degradation in new tissue formation and remodeling phases. The endocytic receptor uPARAP regulates collagen uptake and intracellular degradation. In this study we examined cutaneous wound repair response of uPARAP null (uPARAP-/-) mice. Full thickness wounds were created on dorsal surface of uPARAP-/- or their wildtype littermates. Wound healing evaluation was done by macroscopic observation, histology, gene transcription and biochemical analysis at specific intervals. We found that absence of uPARAP delayed re-epithelialization during wound closure, and altered stiffness of the scar tissue. Despite the absence of the uPARAP-mediated intracellular pathway for collagen degradation, there was no difference in total collagen content of the wounds in uPARAP-/- compared to wildtype mice. This suggests in the absence of uPARAP, a compensatory feedback mechanism functions to keep net collagen in balance.

Cdc42 inhibits ERK-mediated collagenase-1 (MMP-1) expression in collagen-activated human keratinocytes.

Following injury, keratinocytes switch gene expression programs from the one that promotes differentiation to the one that supports migration. A common feature of human wounds and ulcerations of any form is the expression of matrix metalloproteinase 1 (MMP-1; collagenase-1) by leading-edge basal keratinocytes migrating across the dermal or provisional matrix. Induction of MMP-1 occurs by signaling from the α2ß1 integrin in contact with dermal fibrillar type I collagen, and the activity of MMP-1 is required for human keratinocytes to migrate on collagen. Thus, MMP-1 serves a critical role in the repair of damaged human skin. Here, we evaluated the mechanisms controlling MMP-1 expression in primary human keratinocytes from neonatal foreskin and adult female skin. Our results demonstrate that shortly following contact with type I collagen extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase were markedly activated, whereas c-Jun N-terminal kinase (JNK) phosphorylation remained at basal levels. ERK inhibition markedly blocked collagen-stimulated MMP-1 expression in keratinocytes. In contrast, inhibiting p38 or JNK pathways had no effect on MMP-1 production. Moreover, investigating the role of Rho GTPases revealed that Cdc42 attenuates MMP-1 expression by suppressing ERK activity. Thus, our data indicate that injured keratinocytes induce MMP-1 expression through ERK activation, and this process is negatively regulated by Cdc42 activity.

Role of IGF-1 pathway in lung fibroblast activation.

BACKGROUND: IGF-1 is elevated in pulmonary fibrosis and acute lung injury, where fibroblast activation is a prominent feature. We previously demonstrated that blockade of IGF pathway in murine model of lung fibrosis improved outcome and decreased fibrosis. We now expand that study to examine effects of IGF pathway on lung fibroblast behaviors that could contribute to fibrosis. METHODS: We first examined mice that express αSMA promoter upstream of GFP reporter treated with A12, a blocking antibody to IGF-1 receptor, after bleomycin induced lung injury. We then examined the effect of IGF-1 alone, or in combination with the pro-fibrotic cytokine TGFß on expression of markers of myofibroblast activation in vitro, including αSMA, collagen α1, type 1, collagen α1, type III, and TGFß expression. RESULTS: After bleomycin injury, we found decreased number of αSMA-GFP + cells in A12 treated mice, validated by αSMA immunofluorescent staining. We found that IGF-1, alone or in combination with TGF-ß, did not affect αSMA RNA expression, promoter activity, or protein levels when fibroblasts were cultured on stiff substrate. IGF-1 stimulated Col1a1 and Col3a1 expression on stiff substrate. In contrast, IGF-1 treatment on soft substrate resulted in upregulation of αSMA gene and protein expression, as well as Col1a1 and Col3a1 transcripts. In conclusion, IGF-1 stimulates differentiation of fibroblasts into a myofibroblast phenotype in a soft matrix environment and has a modest effect on αSMA stress fiber organization in mouse lung fibroblasts.

PAR1- and PAR2-induced innate immune markers are negatively regulated by PI3K/Akt signaling pathway in oral keratinocytes.

BACKGROUND: Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. RESULTS: Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. CONCLUSION: Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.

Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria.

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.

Modulation of expression of innate immunity markers CXCL5/ENA-78 and CCL20/MIP3alpha by protease-activated receptors (PARs) in human gingival epithelial cells.

Protease-activated receptors (PARs) are G-protein-coupled receptors with an active role in host defense. The two most highly expressed members of the PAR family in gingival epithelial cells (GECs) are PAR1 and PAR2. The major virulence factors of periodontal pathogen Porphyromonas gingivalis are its proteases which can activate PAR2. However, little is known about the function of PARs in GECs when they are activated by their endogenous agonist enzymes. The purpose of this study was to characterize how the expression of innate immune markers is modulated when PAR1 and PAR2 are activated by their agonist enzymes, thrombin and trypsin, respectively. Here, we report that activation of PAR1 and PAR2 induces cell proliferation at low concentration. Activation of PAR via proteolytic activity of thrombin and trypsin induces expression of CXCL5/ENA-78 and CCL20/MIP3alpha in a concentration-dependent manner. Induction of CXCL5 via PAR1 was inhibited in the presence of PAR1 cleavage blocking antibodies and by PAR1 siRNA. The induction of CXCL5 and CCL20 via PAR2 was inhibited by PAR2 siRNA. These findings indicate an active role in innate immune responses by PAR1 and PAR2 in GECs. Modulation of innate immunity by PARs may contribute to co-ordinated and balanced immunosurveillance in GECs.

Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis secretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed to P. gingivalis cell-free culture supernatant or purified RgpB. hBD-2 mRNA in GECs was induced in response to supernatant and purified RgpB from P. gingivalis (P = 0.02 and P = 0.016, respectively). This effect was abrogated by the protease inhibitor tosyl-l-lysine chloromethyl ketone (TLCK) (P < 0.05). In response to P. gingivalis supernatant and to purified RgpB, the hBD-2 mRNA expression was significantly decreased in PAR-2 gene knockdown cells, whereas no change was detected in PAR-1 gene knockdown cells. CCL20 mRNA expression also increased in response to the supernatant of P. gingivalis, and this effect was blocked by the protease inhibitor, TLCK (P = 0.05 and P = 0.024, respectively), and was blocked in PAR-2 gene knockdown cells. Our data indicate that hBD-2 and CCL20 mRNA up-regulation by P. gingivalis supernatant and purified RgpB was mediated via PAR-2, but not via PAR-1, and that proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity.