A review of T helper 17 cell-related cytokines in serum and saliva in periodontitis.

Periodontitis is a chronic inflammatory disease with a complex underlying immunopathology. Cytokines, as molecular mediators of inflammation, play a role in all stages of disease progression. T helper 17 (Th17) cells are thought to play a role in periodontitis. Th17 cell development and maintenance requires a pro-inflammatory cytokine milieu, with many of the cytokines implicated in the pathogenesis of periodontitis. Serum and saliva are easily accessible biofluids which can represent the systemic and local environment to promote the development of Th17 cells. Here we review human clinical studies that investigate IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40L and TNF-α in serum and saliva in periodontitis. We highlight their putative role in the pathogenesis of periodontitis and place them within a wider context of animal and other clinical studies.

Peripheral neutrophil phenotypes during management of periodontitis.

BACKGROUND AND OBJECTIVES: Neutrophils are emerging as a key player in periodontal pathogenesis. The surface expression of cellular markers enables functional phenotyping of neutrophils which have distinct roles in disease states. This study aimed to evaluate the effect of periodontal management on neutrophil phenotypes in peripheral blood in periodontitis patients over one year. MATERIALS AND METHODS: Peripheral blood and the periodontal parameters, mean probing depth and percentage of sites with bleeding on probing (%BOP), were collected from 40 healthy controls and 54 periodontitis patients at baseline and 3-, 6- and 12- months post-treatment. Flow cytometry was used to identify CD11b+ , CD16b+ , CD62L- and CD66b+ expression on neutrophils, neutrophil maturation stages as promyelocytes (CD11b- CD16b- ), metamyelocytes (CD11b+ CD16b- ) and mature neutrophils (CD11b+ CD16b+ ), and suppressive neutrophil phenotype as bands (CD16dim CD62Lbright ), normal neutrophils (CD16bright CD62Lbright ) and suppressive neutrophils (CD16bright CD62Ldim ). RESULTS: CD62L- expression decreased with treatment. No differences were observed in neutrophil maturation stages in health or disease upon treatment. Suppressive and normal neutrophils showed a reciprocal relationship, where suppressive neutrophils decreased with treatment and normal neutrophils increased with treatment. In addition, %BOP was associated with suppressive neutrophils. CONCLUSION: This study demonstrates that management of periodontitis significantly modifies distinct neutrophil phenotypes in peripheral blood. Suppressive neutrophils may play a role in the pathogenesis of periodontitis. However, their exact role is unclear and requires further investigation.

Peripheral T helper cell profiles during management of periodontitis.

AIM: Periodontitis has been associated with other systemic diseases with underlying inflammation responsible for the shared link. This study evaluated longitudinal variation in peripheral T helper cells in periodontitis patients undergoing management over 1 year. MATERIALS AND METHODS: Periodontal parameters and peripheral blood mononuclear cells (PBMCs) were collected from 54 periodontitis patients at baseline, and 3-, 6- and 12-months post-treatment and 40 healthy controls. IFN-γ+ , IL-4+ , IL-17+ and Foxp3+ and their double-positive expression were identified in CD4+ and TCRαß+ cells using flow cytometry. PBMCs were incubated with P. gingivalis, and IFN-γ, IL-4, IL-17 and IL-10 in cell supernatant were measured by ELISA. Cells and cytokines were also assessed based on clinical response to treatment where good (<10% of sites), moderate (10-20%) and poor (>20%) treatment outcome (TxO) groups had probing depths of ≥5 mm at study conclusion. RESULTS: IFN-γ+ cells were lower at baseline, and 3- and 6-months compared to health, whereas Foxp3+ cells were increased at 12-months compared to all preceding timepoints and health. The good TxO group showed treatment-related variation in IFN-γ+ and Foxp3+ cells, whereas the poor TxO group did not. IFN-γ and IL-17 cytokine expression in cell supernatants was significantly lower at baseline compared to health, and IFN-γ and IL-10 showed treatment-related decrease. CONCLUSION: This study suggests that IFN-γ+ and Foxp3+ cells may have a role in the systemic compartment in periodontitis. Periodontal management has local and systemic effects, and thus, assessment and management of periodontitis should form an integral part of overall systemic health.

Peripheral memory T-cell profile is modified in patients undergoing periodontal management.

AIMS: T-cells are known to have a role in periodontitis, however, the effect of periodontal therapy on peripheral memory T-cells is unclear. This study evaluated variation in peripheral memory T-cells and red complex bacteria in sub-gingival plaque in patients undergoing periodontal management. METHODS: Peripheral blood mononuclear cells and sub-gingival plaque were collected from 54 periodontitis patients at baseline, 3-, 6- and 12-months post-therapy and 40 healthy controls. Periodontitis patients were divided into treatment outcome (TxO) groups based on prevalence of sites with probing depth ≥5 mm as good (<10% of sites), moderate (10-20%) or poor (>20%) at study conclusion. Naïve (TN -CCR7+ CD45RA+ ), central memory (TCM -CCR7+ CD45RA- ), effector memory (TEM -CCR7- CD45RA- ) and effector memory T-cells re-expressing CD45RA (TEMRA -CCR7- CD45RA+ ) were phenotyped using flow cytometry in CD4+ , CD8+ , CD4+ CD8+ and CD4- CD8- T-cells and red complex bacteria were quantified using qPCR. RESULTS: At baseline, periodontitis subjects had significantly greater mean probing depths and Porphyromonas gingivalis proportions, lower TN but higher CD4+ TCM , CD8+ TCM , CD4+ CD8+ TEM and CD4- CD8- TEM cell proportions compared to health. Periodontal therapy decreased mean probing depths, P. gingivalis proportions, TEM and CD4+ and CD8+ TCM cells, but increased TN and CD4+ and CD8+ TEMRA cells. The T-cell profile in the good TxO group showed therapy-related changes in CD4+ TEM , and CD8+ TN and TEM cells, whereas, no changes were observed in the poor TxO group. CONCLUSION: Management and the reduction in red complex bacteria were associated with changes in peripheral memory T-cells in periodontitis.

T helper 17 cell-related cytokines in serum and saliva during management of periodontitis.

AIM: T helper (Th)17 cells are implicated in the pathogenesis of periodontitis. This study investigated the effect of periodontal management on fifteen Th17-related cytokines in serum and saliva in periodontitis patients. MATERIALS AND METHODS: Periodontal parameters, serum and saliva were collected from 40 healthy controls and 54 periodontitis subjects before treatment, and 3-, 6- and 12-months post-treatment. Cytokine concentrations of IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40L and TNF-α were determined by Luminex assay. RESULTS: IL-1ß, IL-6, sCD40L and TNF-α in serum, and IL-1ß, IL-6, IL-25 and IL-31 in saliva were significantly higher at baseline compared to health and decreased with treatment. In contrast, serum IL-31 was significantly lower at baseline compared to health and increased with treatment. In addition, salivary IL-10, IL-17A, IL-17F, IL-23, IL-33, IFN-γ and TNF-α also displayed treatment-related reduction. Correlation networks showed that cytokines in saliva displayed a higher number of correlations compared to serum in periodontitis. CONCLUSION: Treatment generally decreased cytokine concentrations except for serum IL-31 which showed a treatment-related increase. Serum cytokine concentrations may not be reflective of salivary cytokines. Saliva may be a better medium for cytokine detection compared to serum. Serum IL-31 and salivary IL-1ß, IL-6, IL-10 and TNF-α were significant predictors for mean probing depth and may be potential biomarkers of interest in the pathogenesis of periodontitis.

Monospecies and polymicrobial biofilms differentially regulate the phenotype of genotype-specific oral cancer cells.

Microbial infection has been shown to involve in oral carcinogenesis; however, the underlying mechanisms remain poorly understood. The present study aimed to characterize the growth of oral microorganisms as both monospecies and polymicrobial biofilms and determine the effects of their products on oral keratinocytes. Candida albicans (ALC3), Actinomyces naeslundii (AN) and Streptococcus mutans (SM) biofilms or a combination of these (TRI) were grown in flow-cell system for 24 h. The biofilms were subjected to fluorescent in situ hybridization using species-specific probes and analysed using confocal laser scanning microscopy. The effluent derived from each biofilm was collected and incubated with malignant (H357) and normal (OKF6) oral keratinocytes to assess extracellular matrix adhesion, epithelial-mesenchymal transition (EMT) and cytokines expression. Incubation of OKF6 with ALC3 and TRI effluent significantly decreased adhesion of the oral keratinocyte to collagen I, whereas incubation of H357 with similar effluent increased adhesion of the oral keratinocyte to laminin I, significantly when compared with incubation with artificial saliva containing serum-free medium (NE; P < 0.05). In OKF6, changes in E-cadherin and vimentin expression were not consistent with EMT although there was evidence of a mesenchymal to epithelial transition in malignant oral keratinocytes incubated with AN and SM effluent. A significant increase of pro-inflammatory cytokines expression, particularly interleukin (IL)-6 and IL-8, was observed when H357 was incubated with all biofilm effluents after 2- and 24-h incubation when compared with NE (P < 0.05). In conclusion, C.albicans, A.naeslundii and S.mutans form polymicrobial biofilms which differentially modulate malignant phenotype of oral keratinocytes.

Porphyromonas gulae Activates Unprimed and Gamma Interferon-Primed Macrophages via the Pattern Recognition Receptors Toll-Like Receptor 2 (TLR2), TLR4, and NOD2.

Porphyromonas gulae is an anaerobic, Gram-negative coccobacillus that has been associated with periodontal disease in companion animals. The aims of this study were to analyze the ligation of pattern recognition receptors by P. gulae and the subsequent activation of macrophages. Exposure of HEK cells transfected with Toll-like receptors (TLRs) or NOD-like receptors to P. gulae resulted in the ligation of TLR2, TLR4, and NOD2. The effects of this engagement of receptors were investigated by measuring the synthesis of nitric oxide (NO), CD86 expression, and inflammatory cytokine production by wild-type, TLR2-/-, and TLR4-/- macrophages. The addition of P. gulae to unprimed and gamma interferon (IFN-γ)-primed (M1 phenotype) macrophages significantly increased the surface expression of CD86, but only M1 macrophages produced nitric oxide. P. gulae-induced expression of CD86 on unprimed macrophages was dependent on both TLR2 and TLR4, but CD86 expression and NO production in M1 macrophages were only TLR2 dependent. P. gulae induced an increase in secretion of interleukin-1α (IL-1α), IL-1ß, IL-6, IL-12p70, IL-13, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) by M1 macrophages compared to that by unprimed controls. Among these cytokines, secretion of IL-6 and TNF-α by M1 macrophages was dependent on either TLR2 or TLR4. Our data indicate that TLR2 and TLR4 are important for P. gulae activation of unprimed macrophages and that activation and effector functions induced in M1 macrophages by P. gulae are mainly dependent on TLR2. In conclusion, P. gulae induces a strong TLR2-dependent inflammatory M1 macrophage response which may be important in establishing the chronic inflammation associated with periodontal disease in companion animals.

Determination of Active Phagocytosis of Unopsonized Porphyromonas gingivalis by Macrophages and Neutrophils Using the pH-Sensitive Fluorescent Dye pHrodo.

Phagocytosis of pathogens is an important component of the innate immune system that is responsible for the removal and degradation of bacteria as well as their presentation via the major histocompatibility complexes to the adaptive immune system. The periodontal pathogen Porphyromonas gingivalis exhibits strain heterogeneity, which may affect a phagocyte's ability to recognize and phagocytose the bacterium. In addition, P. gingivalis is reported to avoid phagocytosis by antibody and complement degradation and by invading phagocytic cells. Previous studies examining phagocytosis have been confounded by both the techniques employed and the potential of the bacteria to invade the cells. In this study, we used a novel, pH-sensitive dye, pHrodo, to label live P. gingivalis strains and examine unopsonized phagocytosis by murine macrophages and neutrophils and human monocytic cells. All host cells examined were able to recognize and phagocytose unopsonized P. gingivalis strains. Macrophages had a preference to phagocytose P. gingivalis strain ATCC 33277 over other strains and clinical isolates in the study, whereas neutrophils favored P. gingivalis W50, ATCC 33277, and one clinical isolate over the other strains. This study revealed that all P. gingivalis strains were capable of being phagocytosed without prior opsonization with antibody or complement.

Porphyromonas gulae Has Virulence and Immunological Characteristics Similar to Those of the Human Periodontal Pathogen Porphyromonas gingivalis.

Periodontitis is a significant problem in companion animals, and yet little is known about the disease-associated microbiota. A major virulence factor for the human periodontal pathogen Porphyromonas gingivalis is the lysyl- and arginyl-specific proteolytic activity of the gingipains. We screened several Porphyromonas species isolated from companion animals-P. asaccharolytica, P. circumdentaria, P. endodontalis, P. levii, P. gulae, P. macacae, P. catoniae, and P. salivosa-for Lys- and Arg-specific proteolytic activity and compared the epithelial and macrophage responses and induction of alveolar bone resorption of the protease active species to that of Porphyromonas gingivalis Only P. gulae exhibited Lys-and Arg-specific proteolytic activity. The genes encoding the gingipains (RgpA/B and Kgp) were identified in the P. gulae strain ATCC 51700 and all publicly available 12 draft genomes of P. gulae strains. P. gulae ATCC 51700 induced levels of alveolar bone resorption in an animal model of periodontitis similar to those in P. gingivalis W50 and exhibited a higher capacity for autoaggregation and binding to oral epithelial cells with induction of apoptosis. Macrophages (RAW 264.7) were found to phagocytose P. gulae ATCC 51700 and the fimbriated P. gingivalis ATCC 33277 at similar levels. In response to P. gulae ATCC 51700, macrophages secreted higher levels of cytokines than those induced by P. gingivalis ATCC 33277 but lower than those induced by P. gingivalis W50, except for the interleukin-6 response. Our results indicate that P. gulae exhibits virulence characteristics similar to those of the human periodontal pathogen P. gingivalis and therefore may play a key role in the development of periodontitis in companion animals.

Macrophage depletion abates Porphyromonas gingivalis-induced alveolar bone resorption in mice.

The role of the macrophage in the immunopathology of periodontitis has not been well defined. In this study, we show that intraoral inoculation of mice with Porphyromonas gingivalis resulted in infection, alveolar bone resorption, and a significant increase in F4/80(+) macrophages in gingival and submandibular lymph node tissues. Macrophage depletion using clodronate-liposomes resulted in a significant reduction in F4/80(+) macrophage infiltration of gingival and submandibular lymph node tissues and significantly (p < 0.01) less P. gingivalis-induced bone resorption compared with controls in BALB/c and C57BL/6 mice. In both mouse strains, the P. gingivalis-specific IgG Ab subclass and serum cytokine [IL-4, IL-10, IFN-γ, and IL-12 (p70)] responses were significantly (p < 0.01) lower in the macrophage-depleted groups. Macrophage depletion resulted in a significant reduction in the level of P. gingivalis infection, and the level of P. gingivalis infection was significantly correlated with the level of alveolar bone resorption. M1 macrophages (CD86(+)), rather than M2 macrophages (CD206(+)), were the dominant macrophage phenotype of the gingival infiltrate in response to P. gingivalis infection. P. gingivalis induced a significant (p < 0.01) increase in NO production and a small increase in urea concentration, as well as a significant increase in the secretion of IL-1ß, IL-6, IL-10, IL-12 (p70), eotaxin, G-CSF, GM-CSF, macrophage chemoattractant protein-1, macrophage inflammatory protein-α and -ß, and TNF-α in isolated murine macrophages. In conclusion, P. gingivalis infection induced infiltration of functional/inflammatory M1 macrophages into gingival tissue and alveolar bone resorption. Macrophage depletion reduced P. gingivalis infection and alveolar bone resorption by modulating the host immune response.

Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors.

Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

GM-CSF and uPA are required for Porphyromonas gingivalis-induced alveolar bone loss in a mouse periodontitis model.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and urokinase-type plasminogen activator (uPA) can contribute to the progression of chronic inflammatory diseases with possible involvement of macrophages. In this study, we investigated the role of both GM-CSF and uPA in Porphyromonas gingivalis-induced experimental periodontitis using GM-CSF-/- and uPA-/- mice. Intra-oral inoculation of wild-type (WT) C57BL/6 mice with P. gingivalis resulted in establishment of the pathogen in plaque and a significant increase in alveolar bone resorption. The infected mice also exhibited a CD11b(+) CD86(+) macrophage infiltrate into the gingival tissue, as well as P. gingivalis-specific pro-inflammatory cytokine and predominantly IgG2b antibody responses. In comparison, intra-oral inoculation of P. gingivalis did not induce bone resorption and there was significantly less P. gingivalis recovered from plaque in GM-CSF-/- and uPA-/- mice. Furthermore, P. gingivalis did not induce a macrophage gingival infiltrate or activate isolated peritoneal macrophages from the gene-deficient mice. Pro-inflammatory P. gingivalis-specific T-cell cytokine responses and serum interferon-gamma (IFN-γ) and IgG2b concentrations were significantly lower in GM-CSF-/- mice. In uPA-/- mice, T-cell responses were lower but serum IFN-γ and IgG2b levels were comparable with WT mice levels. These results suggest that GM-CSF and uPA are both involved in the progression of experimental periodontitis, possibly via a macrophage-dependent mechanism(s).

Control of macrophage lineage populations by CSF-1 receptor and GM-CSF in homeostasis and inflammation.

There is recent interest in the role of monocyte/macrophage subpopulations in pathology. How the hemopoietic growth factors, macrophage-colony stimulating factor (M-CSF or CSF-1) and granulocyte macrophage (GM)-CSF, regulate their in vivo development and function is unclear. A comparison is made here on the effect of CSF-1 receptor (CSF-1R) and GM-CSF blockade/depletion on such subpopulations, both in the steady state and during inflammation. In the steady state, administration of neutralizing anti-CSF-1R monoclonal antibody (mAb) rapidly (within 3-4 days) lowered, specifically, the number of the more mature Ly6C(lo) peripheral blood murine monocyte population and resident peritoneal macrophages; it also reduced the accumulation of murine exudate (Ly6C(lo)) macrophages in two peritonitis models and alveolar macrophages in lung inflammation, consistent with a non-redundant role for CSF-1 (or interleukin-34) in certain inflammatory reactions. A neutralizing mAb to GM-CSF also reduced inflammatory macrophage numbers during antigen-induced peritonitis and lung inflammation. In GM-CSF gene-deficient mice, a detailed kinetic analysis of monocyte/macrophage and neutrophil dynamics in antigen-induced peritonitis suggested that GM-CSF was acting, in part, systemically to maintain the inflammatory reaction. A model is proposed in which CSF-1R signaling controls the development of the macrophage lineage at a relatively late stage under steady state conditions and during certain inflammatory reactions, whereas in inflammation, GM-CSF can be required to maintain the response by contributing to the prolonged extravasation of immature monocytes and neutrophils. A correlation has been observed between macrophage numbers and the severity of certain inflammatory conditions, and it could be that CSF-1 and GM-CSF contribute to the control of these numbers in the ways proposed.

Sensitizing and Th2 adjuvant activity of cysteine protease allergens.

BACKGROUND: Innate properties that enhance immune responses might increase the propensity of certain allergens to induce allergic sensitization. Either a direct adjuvant effect or the increased immune response to the allergen could then increase allergic responses to bystander antigens. Here, we report on a model that does not use Th2-skewing adjuvants and yet achieves sensitization solely via the nasal mucosa. METHODS: Animals were sensitized with either enzymatically active, inactive or non-activated cysteine proteases via the nasal mucosa. Following two sensitization phases, mice were challenged with a higher dose of allergen. For bystander sensitization, mice received recombinant Der p 2 at sensitization in conjunction with the cysteine protease and were challenged with rDer p 2 alone. Sensitization was determined by measuring allergen-specific antibody responses and cytokine and cellular infiltrates into the lungs following challenge. RESULTS: Sensitization for Th2-type lung hypersensitivity for both the cysteine protease and bystander antigens was readily achieved and both were dependent on the proteolytic activity of the allergen. Bystander adjuvant activity was demonstrated for mice that were low IgE responders to the cysteine protease, showing a response independent from the immune response to the enhancing cysteine protease. Airway hyperreactivity was induced in the susceptible NOD strain of mouse, and mice subjected to prolonged administration of papain maintained the ability to produce lung hypersensitivity and Th2-type responses. CONCLUSIONS: These experiments demonstrate that cysteine protease activity at low doses can be an adjuvant for respiratory Th2 responses for themselves and bystander antigens in the absence of another adjuvant.

Regulation of systemic and local myeloid cell subpopulations by bone marrow cell-derived granulocyte-macrophage colony-stimulating factor in experimental inflammatory arthritis.

OBJECTIVE: Even though there are clinical trials assessing granulocyte-macrophage colony-stimulating factor (GM-CSF) blockade in rheumatoid arthritis (RA), questions remain as to how GM-CSF acts as a proinflammatory cytokine. The aims of this study on the regulation of arthritis progression by GM-CSF were to determine the source of the GM-CSF, whether there are systemic effects, the changes in synovial tissue leukocyte populations, and the arthritis model dependence on GM-CSF. METHODS: Bone marrow chimeras were used to determine the source of GM-CSF required for the development of collagen-induced arthritis (CIA). The K/BxN serum-transfer model of arthritis was tested in GM-CSF(-/-) mice and using anti-GM-CSF monoclonal antibodies. Cell populations from arthritic mice were assessed by differential staining and flow cytometry. RESULTS: In the CIA model, GM-CSF produced by bone marrow-derived cells was required for arthritis development. GM-CSF blockade, while ameliorating the development of CIA, was found to have systemic effects, limiting the increase in circulating Ly-6C(high) monocytes and neutrophils. GM-CSF blockade led to fewer synovial macrophages (both Ly-6C(high) and Ly-6C(low)), neutrophils, and lymphocytes. In the absence of GM-CSF, K/BxN serum-transfer arthritis initially developed normally; however, the numbers of Ly-6C(high) monocytes and synovial macrophages (both Ly-6C(high) and Ly-6C(low)) were again reduced, along with the peak disease severity and maintenance. CONCLUSION: GM-CSF is a key player in two arthritis models, participating in interactions between hemopoietic cells, both locally and systemically, to control myeloid cell numbers as well as presumably to "activate" them. These results could be useful for the analysis of current clinical trials targeting GM-CSF in patients with RA.

The Potential of Modified and Multimeric Antimicrobial Peptide Materials as Superbug Killers.

Antimicrobial peptides (AMPs) are found in nearly all living organisms, show broad spectrum antibacterial activity, and can modulate the immune system. Furthermore, they have a very low level of resistance induction in bacteria, which makes them an ideal target for drug development and for targeting multi-drug resistant bacteria 'Superbugs'. Despite this promise, AMP therapeutic use is hampered as typically they are toxic to mammalian cells, less active under physiological conditions and are susceptible to proteolytic degradation. Research has focused on addressing these limitations by modifying natural AMP sequences by including e.g., d-amino acids and N-terminal and amino acid side chain modifications to alter structure, hydrophobicity, amphipathicity, and charge of the AMP to improve antimicrobial activity and specificity and at the same time reduce mammalian cell toxicity. Recently, multimerisation (dimers, oligomer conjugates, dendrimers, polymers and self-assembly) of natural and modified AMPs has further been used to address these limitations and has created compounds that have improved activity and biocompatibility compared to their linear counterparts. This review investigates how modifying and multimerising AMPs impacts their activity against bacteria in planktonic and biofilm states of growth.

Tumor Associated Macrophages: Origin, Recruitment, Phenotypic Diversity, and Targeting.

The tumor microenvironment (TME) is known to have a strong influence on tumorigenesis, with various components being involved in tumor suppression and tumor growth. A protumorigenic TME is characterized by an increased infiltration of tumor associated macrophages (TAMs), where their presence is strongly associated with tumor progression, therapy resistance, and poor survival rates. This association between the increased TAMs and poor therapeutic outcomes are stemming an increasing interest in investigating TAMs as a potential therapeutic target in cancer treatment. Prominent mechanisms in targeting TAMs include: blocking recruitment, stimulating repolarization, and depletion methods. For enhancing targeting specificity multiple nanomaterials are currently being explored for the precise delivery of chemotherapeutic cargo, including the conjugation with TAM-targeting peptides. In this paper, we provide a focused literature review of macrophage biology in relation to their role in tumorigenesis. First, we discuss the origin, recruitment mechanisms, and phenotypic diversity of TAMs based on recent investigations in the literature. Then the paper provides a detailed review on the current methods of targeting TAMs, including the use of nanomaterials as novel cancer therapeutics.

Architectural Effects of Star-Shaped "Structurally Nanoengineered Antimicrobial Peptide Polymers" (SNAPPs) on Their Biological Activity.

In this work, the effect of two key structural parameters, number of arms and arm length, of star-shaped "structurally nanoengineered antimicrobial peptide polymers" (SNAPPs) on their antimicrobial activity and biocompatibility, is investigated. A library of star-shaped SNAPPs is prepared, containing varying arm numbers and arm lengths. Antimicrobial assays are then performed to assess the capacity of the SNAPPs to disrupt the membrane, inhibit the growth, and kill pathogenic bacteria. A major finding of the study is that increasing arm number and length of SNAPPs enhanced antimicrobial activity, which can be respectively attributed to the higher local concentrations of polypeptide arms and increased α-helical content. SNAPP architecture is shown to affect the bacteria membrane state and therefore mechanism of killing. Two more potent structures with up to twice the antimicrobial activity of the previously reported SNAPP are discovered in this process. Toxicities of the SNAPPs also increase with arm number and arm length, however therapeutic index calculations identified a 16-arm SNAPP and an easier to prepare 4-arm SNAPP as the best therapeutic agents. The biocompatibility of the SNAPP with the best biological activity is also evaluated in vivo, showing no markers of systemic damage in mice.

Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo.

Outer membrane vesicles (OMVs) are proteoliposomes blebbed from the surface of Gram-negative bacteria. Chronic periodontitis is associated with an increase in subgingival plaque of Gram-negative bacteria, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. In this study, we investigated the immune-modulatory effects of P. gingivalis, T. denticola, and T. forsythia OMVs on monocytes and differentiated macrophages. All of the bacterial OMVs were phagocytosed by monocytes, M(naïve) and M(IFNγ) macrophages in a dose-dependent manner. They also induced NF-κB activation and increased TNFα, IL-8, and IL-1ß cytokine secretion. P. gingivalis OMVs were also found to induce anti-inflammatory IL-10 secretion. Although unprimed monocytes and macrophages were resistant to OMV-induced cell death, lipopolysaccharide or OMV priming resulted in a significantly reduced cell viability. P. gingivalis, T. denticola, and T. forsythia OMVs all activated inflammasome complexes, as monitored by IL-1ß secretion and ASC speck formation. ASC was critical for OMV-induced inflammasome formation, while AIM2-/- and Caspase-1-/- cells had significantly reduced inflammasome formation and NLRP3-/- cells exhibited a slight reduction. OMVs were also found to provide both priming and activation of the inflammasome complex. High-resolution microscopy and flow cytometry showed that P. gingivalis OMVs primed and activated macrophage inflammasomes in vivo with 80% of macrophages exhibiting inflammasome complex formation. In conclusion, periodontal pathogen OMVs were found to have significant immunomodulatory effects upon monocytes and macrophages and should therefore influence pro-inflammatory host responses associated with disease.

The effect of tissue type-plasminogen activator deletion and associated fibrin(ogen) deposition on macrophage localization in peritoneal inflammation.

There are two plasminogen activators (PAs), urokinase type-PA (u-PA) and tissue type-PA (t-PA). While u-PA is considered to be involved in cellular migration and tissue remodeling and t-PA in fibrinolysis, this distinction is not always clear-cut. With the use of u-PA and t-PA gene deficient mice (u-PA-/- and t-PA-/- mice, respectively) we have assessed the role of each PA in acute peritonitis. The cellular infiltrate in both thioglycolate- and antigen-induced peritoneal exudates was unaffected in u-PA-/- mice; in contrast, in t-PA-/- mice, the macrophage numbers, particularly of the Mac-1(hi) population, in the peritoneal cavity by day 4 were significantly reduced compared to wild-type mice. However, examination of the peritoneal wall revealed in fact increased numbers of macrophages adhering on/in the cavity lining at all time points studied; in addition, increased fibrin(ogen) staining was observed for these mice. The reduced macrophage numbers in the peritoneal cavities of t-PA-/- mice could be increased by administration of plasmin or t-PA prior to harvesting the thioglycolate-elicited exudates. These results suggest that t-PA and not u-PA is the PA controlling fibrinolysis in murine peritonitis. In its absence macrophages adhere to the accumulated fibrin(ogen) on/in the cavity wall lining, most likely via Mac-1 binding, thus affecting migration into and/or out of the peritoneal cavity. They also highlight the need to examine both the peritoneal cavity and wall in order to monitor accurately the extent of a peritoneal inflammatory reaction. Peritoneal inflammation in t-PA-/- mice represents a useful model to study the progression of intra-abdominal adhesions during surgery and clinical peritonitis.